Biosensors for biological or chemical analysis and systems and methods for same

ABSTRACT

A biosensor is provided including a detection device and a flow cell mounted to the detection device. The detection device has a detector surface with a plurality of reaction sites. The detection device also includes a filter layer that is configured to at least one of (a) filter unwanted excitation light signals; (b) direct emission signals from a designated reaction site toward one or more associated light detectors that are configured to detect the emission signals from the designated reaction site; or (c) block or prevent detection of crosstalk emission signals from adjacent reaction sites.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/995,767, filed on Jun. 1, 2018, which is a continuation of U.S.application Ser. No. 14/552,673, filed on Nov. 25, 2014, now U.S. Pat.No. 9,990,381, which is a divisional of U.S. application Ser. No.13/833,619, filed on Mar. 15, 2013, now U.S. Pat. No. 8,906,320, whichclaims the benefit of U.S. Provisional Application No. 61/625,051, filedon Apr. 16, 2012, all of which are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

Embodiments of the present invention relate generally to biological orchemical analysis and more particularly to systems and methods usingdetection devices for biological or chemical analysis.

Various protocols in biological or chemical research involve performinga large number of controlled reactions on local support surfaces orwithin predefined reaction chambers. The desired reactions may then beobserved or detected and subsequent analysis may help identify or revealproperties of chemicals involved in the reaction. For example, in somemultiplex assays, an unknown analyte having an identifiable label (e.g.,fluorescent label) may be exposed to thousands of known probes undercontrolled conditions. Each known probe may be deposited into acorresponding well of a microplate. Observing any chemical reactionsthat occur between the known probes and the unknown analyte within thewells may help identify or reveal properties of the analyte. Otherexamples of such protocols include known DNA sequencing processes, suchas sequencing-by-synthesis (SBS) or cyclic-array sequencing.

In some conventional fluorescent-detection protocols, an optical systemis used to direct an excitation light onto fluorescently-labeledanalytes and to also detect the fluorescent signals that may emit fromthe analytes. However, such optical systems can be relatively expensiveand require a larger benchtop footprint. For example, the optical systemmay include an arrangement of lenses, filters, and light sources. Inother proposed detection systems, the controlled reactions occurimmediately over a solid-state imager (e.g., charged-coupled device(CCD) or a complementary metal-oxide-semiconductor (CMOS) detector) thatdoes not require a large optical assembly to detect the fluorescentemissions.

However, the proposed solid-state imaging systems may have somelimitations. For example, it may be challenging to distinguish thefluorescent emissions from the excitation light when the excitationlight is also directed toward the light detectors of the solid-stateimager. In addition, fluidicly delivering reagents to analytes that arelocated on an electronic device and in a controlled manner may presentadditional challenges. As another example, fluorescent emissions aresubstantially isotropic. As the density of the analytes on thesolid-state imager increases, it becomes increasingly challenging tomanage or account for unwanted light emissions from adjacent analytes(e.g., crosstalk).

BRIEF DESCRIPTION OF THE INVENTION

In accordance with one embodiment, a biosensor is provided that includesa flow cell mounted to a detection device. The detection device has adetector surface with a plurality of reaction sites. The detectiondevice includes a filter layer that is configured to at least one of (a)filter unwanted light signals, such as light signals from excitationlight; (b) direct emission signals from a designated reaction sitetoward one or more detectors that are configured to detect the emissionsignals from the designated reaction site; or (c) block or preventdetection of emission signals from adjacent reaction sites.

In another embodiment, a biosensor is provided that includes a flow celland a detection device having a plurality of stacked layers and adetector surface configured to support reaction sites. The stackedlayers include a filter layer and a solid-state imager coupled to thefilter layer. The filter layer includes filter walls and alight-absorbing material that is deposited between adjacent filterwalls. The light-absorbing material configured to prevent transmissionof excitation signals and permit transmission of fluorescent signals.Adjacent filter walls define a detection path therebetween through thecorresponding light-absorbing material toward the solid-state imager.The filter walls are configured to reflect the fluorescent signals. Theflow cell is mounted to the detector surface thereby defining a flowchannel between at least one surface of the flow cell (e.g. an exteriorsurface of the flow cell) and the detection device. The surface of theflow cell can includes a material that permits transmission of theexcitation signals. Thus, the surface can function to retain fluid inthe flow channel while passing excitation light to the fluid.

In another embodiment, a method of analyzing fluorescent signalsdetected from an array of reaction sites having correspondinganalytes-of-interest. The method includes performing first and secondimaging events using a detection device (e.g., CMOS imager) having anarray of light detectors in which (a) fluorescent signals emitting froma first set of reaction sites are captured during the first imagingevent and (b) fluorescent signals emitting from a different second setof reaction sites are captured during the second imaging event. For eachof the first and second imaging events, the light detectors have a lightscore that corresponds to an amount of fluorescence detected during thecorresponding imaging event. The method can also include identifying anamount of crosstalk that is detected by a first light detector when anadjacent second light detector detects a positive binding event. Themethod can also include analyzing the light scores from the first andsecond imaging events to determine information about theanalytes-of-interest, wherein the analyzing includes accounting for thecrosstalk detected by light detectors.

In yet another embodiment, a method of detecting fluorescent signalsfrom an array of reaction sites that are distributed along a detectorsurface of a detection device is provided. The method includes providingthe detection device including the detector surface and a solid-stateimager having light detectors. The detection device also includes filterwalls that extend between and define detection paths between thedetector surface and associated light detectors of the solid-stateimager. The detection paths include first and second detection paths,and the light detectors include first and second light detectors thatdetect light signals propagating along the first and second detectionpaths, respectively. The first and second detection paths are adjacentto each other and the first and second light detectors are adjacent toeach other. The method can also include, during a first imaging event,detecting a positive portion of fluorescent signals from a firstreaction site on the detector surface with the first light detector. Thepositive portion indicates that a desired reaction has occurred at thefirst reaction site. The method can also include, during the firstimaging event, detecting a crosstalk portion of the fluorescent signalsfrom the first reaction site with the second light detector, wherein thecrosstalk portion is less than the positive portion. The method can alsoinclude, during a second imaging event, detecting a positive portion offluorescent signals from a second reaction site on the detector surfacewith the second light detector. The positive portion indicates that adesired reaction has occurred at the second reaction site. The methodcan also include analyzing the detected fluorescent signals to determineinformation about analytes-of-interest at the first and second reactionsites. The analyzing optionally includes accounting for the crosstalkportion.

In another embodiment, a method of analyzing signal data from abiosensor including a detection device is provided. The detection deviceincludes an array of light detectors. Each of the light detectors isassociated with at least one reaction site. The reaction sites includeanalytes-of-interest. The method includes obtaining the signal data fromthe light detectors. The signal data includes light scores that arebased on an amount of light detected by the light detectors during aplurality of imaging events. The method also includes analyzing thelight scores from a set of light detectors for each of the plurality ofthe imaging events. The method also includes determining respectivecrosstalk functions of the light detectors in the set in which each ofthe crosstalk functions is based on an amount of light detected by otherlight detectors in the set. The method also includes analyzing thesignal data for each of the imaging events using the crosstalk functionsto determine characteristics of the analytes-of-interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of an exemplary system for biological orchemical analysis formed in accordance with one embodiment.

FIG. 2 is a block diagram of an exemplary system controller that may beused in the system of FIG. 1.

FIG. 3 is a block diagram of an exemplary workstation for biological orchemical analysis in accordance with one embodiment.

FIG. 4 is a perspective view of an exemplary workstation and anexemplary cartridge in accordance with one embodiment.

FIG. 5 is a front view of an exemplary rack assembly that includes aplurality of the workstations of FIG. 4.

FIG. 6 illustrates internal components of an exemplary cartridge.

FIG. 7 illustrates a cross-section of an exemplary biosensor formed inaccordance with one embodiment.

FIG. 8 is a flowchart illustrating an exemplary method of manufacturingthe biosensor of FIG. 7.

FIGS. 9-16 illustrate the biosensor of FIG. 7 at different manufacturingstages.

FIG. 17 is a diagrammatical representation of general phases inpatterning a detector surface of an exemplary biosensor.

FIGS. 18-23 are diagrammatical representations of successive steps inthe disposition of sites on a detector surface of an exemplarybiosensor.

FIGS. 24-26 are diagrammatical representations of steps in thepreparation of reaction sites.

FIGS. 27 and 28 are diagrammatical representations of single nucleicacid molecule capture followed by amplification to create multiplecopies of the nucleic acid molecule.

FIG. 29 is an enlarged cross-section of a filter layer that may be usedin a biosensor formed in accordance with one embodiment.

FIG. 30 illustrates an exemplary detection device for biological and/orchemical analysis formed in accordance with one embodiment.

FIG. 31 is a top plan view of an exemplary detector surface duringfirst, second, and third imaging events in accordance with oneembodiment.

FIG. 32 illustrates light scores or values obtained by light detectorsin an exemplary detection device in accordance with one embodiment.

FIG. 33 shows an exemplary method of analyzing signal data obtained froma detection device used for biological and/or chemical analysis.

FIG. 34 is a top plan view of an exemplary detector surface having aplurality of reaction sites in accordance with one embodiment.

FIG. 35 shows plan views of various configurations of flow cells thatmay be used with one or more embodiments.

FIG. 36 illustrates a cross-section of an exemplary biosensor formed inaccordance with one embodiment.

FIG. 37 shows mismatches on a cycle by cycle basis from a sequencingrun, or one operational session.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments described herein may be used in various biological orchemical processes and systems for academic or commercial analysis. Morespecifically, embodiments described herein may be used in variousprocesses and systems where it is desired to detect an event, property,quality, or characteristic that is indicative of a desired reaction. Forexample, embodiments described herein include cartridges, biosensors,and their components as well as bioassay systems that operate withcartridges and biosensors. In particular embodiments, the cartridges andbiosensors include a flow cell and one or more light detectors that arecoupled together in a substantially unitary structure.

The bioassay systems may be configured to perform a plurality of desiredreactions that may be detected individually or collectively. Thebiosensors and bioassay systems may be configured to perform numerouscycles in which the plurality of desired reactions occurs in parallel.For example, the bioassay systems may be used to sequence a dense arrayof DNA features through iterative cycles of enzymatic manipulation andimage acquisition. As such, the cartridges and biosensors may includeone or more microfluidic channels that deliver reagents or otherreaction components to a reaction site. In some embodiments, thereaction sites are randomly distributed across a substantially planersurface. For example, the reaction sites may have an uneven distributionin which some reaction sites are located closer to each other than otherreaction sites. In other embodiments, the reaction sites are patternedacross a substantially planer surface in a predetermined manner. Each ofthe reaction sites may be associated with one or more light detectorsthat detect light from the associated reaction site. Yet in otherembodiments, the reaction sites are located in reaction chambers thatcompartmentalize the desired reactions therein.

The following detailed description of certain embodiments will be betterunderstood when read in conjunction with the appended drawings. To theextent that the figures illustrate diagrams of the functional blocks ofvarious embodiments, the functional blocks are not necessarilyindicative of the division between hardware circuitry. Thus, forexample, one or more of the functional blocks (e.g., processors ormemories) may be implemented in a single piece of hardware (e.g., ageneral purpose signal processor or random access memory, hard disk, orthe like). Similarly, the programs may be stand alone programs, may beincorporated as subroutines in an operating system, may be functions inan installed software package, and the like. It should be understoodthat the various embodiments are not limited to the arrangements andinstrumentality shown in the drawings.

As used herein, an element or step recited in the singular and proceededwith the word “a” or “an” should be understood as not excluding pluralof said elements or steps, unless such exclusion is explicitly stated.Furthermore, references to “one embodiment” are not intended to beinterpreted as excluding the existence of additional embodiments thatalso incorporate the recited features. Moreover, unless explicitlystated to the contrary, embodiments “comprising” or “having” an elementor a plurality of elements having a particular property may includeadditional elements whether or not they have that property.

As used herein, a “desired reaction” includes a change in at least oneof a chemical, electrical, physical, or optical property (or quality) ofan analyte-of-interest. In particular embodiments, the desired reactionis a positive binding event (e.g., incorporation of a fluorescentlylabeled biomolecule with the analyte-of-interest). More generally, thedesired reaction may be a chemical transformation, chemical change, orchemical interaction. The desired reaction may also be a change inelectrical properties. For example, the desired reaction may be a changein ion concentration within a solution. Exemplary reactions include, butare not limited to, chemical reactions such as reduction, oxidation,addition, elimination, rearrangement, esterification, amidation,etherification, cyclization, or substitution; binding interactions inwhich a first chemical binds to a second chemical; dissociationreactions in which two or more chemicals detach from each other;fluorescence; luminescence; bioluminescence;

-   -   chemiluminescence; and biological reactions, such as nucleic        acid replication, nucleic acid amplification, nucleic acid        hybridization, nucleic acid ligation, phosphorylation, enzymatic        catalysis, receptor binding, or ligand binding. The desired        reaction can also be addition or elimination of a proton, for        example, detectable as a change in pH of a surrounding solution        or environment. An additional desired reaction can be detecting        the flow of ions across a membrane (e.g., natural or synthetic        bilayer membrane), for example as ions flow through a membrane        the current is disrupted and the disruption can be detected.

In particular embodiments, the desired reaction includes theincorporation of a fluorescently-labeled molecule to an analyte. Theanalyte may be an oligonucleotide and the fluorescently-labeled moleculemay be a nucleotide. The desired reaction may be detected when anexcitation light is directed toward the oligonucleotide having thelabeled nucleotide, and the fluorophore emits a detectable fluorescentsignal. In alternative embodiments, the detected fluorescence is aresult of chemiluminescence or bioluminescence. A desired reaction mayalso increase fluorescence (or Förster) resonance energy transfer(FRET), for example, by bringing a donor fluorophore in proximity to anacceptor fluorophore, decrease FRET by separating donor and acceptorfluorophores, increase fluorescence by separating a quencher from afluorophore or decrease fluorescence by co-locating a quencher andfluorophore.

As used herein, a “reaction component” or “reactant” includes anysubstance that may be used to obtain a desired reaction. For example,reaction components include reagents, enzymes, samples, otherbiomolecules, and buffer solutions. The reaction components aretypically delivered to a reaction site in a solution and/or immobilizedat a reaction site. The reaction components may interact directly orindirectly with another substance, such as the analyte-of-interest.

As used herein, the term “reaction site” is a localized region where adesired reaction may occur. A reaction site may include support surfacesof a substrate where a substance may be immobilized thereon. Forexample, a reaction site may include a substantially planar surface in achannel of a flow cell that has a colony of nucleic acids thereon.Typically, but not always, the nucleic acids in the colony have the samesequence, being for example, clonal copies of a single stranded ordouble stranded template. However, in some embodiments a reaction sitemay contain only a single nucleic acid molecule, for example, in asingle stranded or double stranded form. Furthermore, a plurality ofreaction sites may be randomly distributed along the support surface orarranged in a predetermined manner (e.g., side-by-side in a matrix, suchas in microarrays). A reaction site can also include a reaction chamberthat at least partially defines a spatial region or volume configured tocompartmentalize the desired reaction. As used herein, the term“reaction chamber” includes a spatial region that is in fluidcommunication with a flow channel. The reaction chamber may be at leastpartially separated from the surrounding environment or other spatialregions. For example, a plurality of reaction chambers may be separatedfrom each other by shared walls. As a more specific example, thereaction chamber may include a cavity defined by interior surfaces of awell and have an opening or aperture so that the cavity may be in fluidcommunication with a flow channel. Biosensors including such reactionchambers are described in greater detail in international applicationno. PCT/US2011/057111, filed on Oct. 20, 2011, which is incorporatedherein by reference in its entirety.

In some embodiments, the reaction chambers are sized and shaped relativeto solids (including semi-solids) so that the solids may be inserted,fully or partially, therein. For example, the reaction chamber may besized and shaped to accommodate only one capture bead. The capture beadmay have clonally amplified DNA or other substances thereon.Alternatively, the reaction chamber may be sized and shaped to receivean approximate number of beads or solid substrates. As another example,the reaction chambers may also be filled with a porous gel or substancethat is configured to control diffusion or filter fluids that may flowinto the reaction chamber.

In some embodiments, light detectors (e.g., photodiodes) are associatedwith corresponding reaction sites. A light detector that is associatedwith a reaction site is configured to detect light emissions from theassociated reaction site when a desired reaction has occurred at theassociated reaction site. In some cases, a plurality of light detectors(e.g. several pixels of a camera device) may be associated with a singlereaction site. In other cases, a single light detector (e.g. a singlepixel) may be associated with a single reaction site or with a group ofreaction sites. The light detector, the reaction site, and otherfeatures of the biosensor may be configured so that at least some of thelight is directly detected by the light detector without beingreflected.

As used herein, the term “adjacent” when used with respect to tworeaction sites means no other reaction site is located between the tworeaction sites. The term “adjacent” may have a similar meaning when usedwith respect to adjacent detection paths and adjacent light detectors(e.g., adjacent light detectors have no other light detectortherebetween). In some cases, a reaction site may not be adjacent toanother reaction site, but may still be within an immediate vicinity ofthe other reaction site. A first reaction site may be in the immediatevicinity of a second reaction site when fluorescent emission signalsfrom the first reaction site are detected by the light detectorassociated with the second reaction site. More specifically, a firstreaction site may be in the immediate vicinity of a second reaction sitewhen the light detector associated with the second reaction sitedetects, for example crosstalk from the first reaction site. Adjacentreaction sites can be contiguous such that they abut each other or theadjacent sites can be non-contiguous having an intervening spacebetween.

As used herein, a “substance” includes items or solids, such as capturebeads, as well as biological or chemical substances. As used herein, a“biological or chemical substance” includes biomolecules,samples-of-interest, analytes-of-interest, and other chemicalcompound(s). A biological or chemical substance may be used to detect,identify, or analyze other chemical compound(s), or function asintermediaries to study or analyze other chemical compound(s). Inparticular embodiments, the biological or chemical substances include abiomolecule. As used herein, a “biomolecule” includes at least one of abiopolymer, nucleoside, nucleic acid, polynucleotide, oligonucleotide,protein, enzyme, polypeptide, antibody, antigen, ligand, receptor,polysaccharide, carbohydrate, polyphosphate, cell, tissue, organism, orfragment thereof or any other biologically active chemical compound(s)such as analogs or mimetics of the aforementioned species.

In a further example, a biological or chemical substance or abiomolecule includes an enzyme or reagent used in a coupled reaction todetect the product of another reaction such as an enzyme or reagent usedto detect pyrophosphate in a pyrosequencing reaction. Enzymes andreagents useful for pyrophosphate detection are described, for example,in U.S. Patent Publication No. 2005/0244870 A1, which is incorporatedherein in its entirety.

Biomolecules, samples, and biological or chemical substances may benaturally occurring or synthetic and may be suspended in a solution ormixture within a spatial region. Biomolecules, samples, and biologicalor chemical substances may also be bound to a solid phase or gelmaterial. Biomolecules, samples, and biological or chemical substancesmay also include a pharmaceutical composition. In some cases,biomolecules, samples, and biological or chemical substances of interestmay be referred to as targets, probes, or analytes.

As used herein, a “biosensor” includes a structure having a plurality ofreaction sites. A biosensor may include a solid-state imaging device(e.g., CCD or CMOS imager) and, optionally, a flow cell mounted thereto.The flow cell may include at least one flow channel that is in fluidcommunication with the reaction sites. As one specific example, thebiosensor is configured to fluidicly and electrically couple to abioassay system. The bioassay system may deliver reactants to thereaction sites according to a predetermined protocol (e.g.,sequencing-by-synthesis) and perform a plurality of imaging events. Forexample, the bioassay system may direct solutions to flow along thereaction sites. At least one of the solutions may include four types ofnucleotides having the same or different fluorescent labels. Thenucleotides may bind to corresponding oligonucleotides located at thereaction sites. The bioassay system may then illuminate the reactionsites using an excitation light source (e.g., solid-state light sources,such as light-emitting diodes or LEDs). The excitation light may have apredetermined wavelength or wavelengths, including a range ofwavelengths. The excited fluorescent labels provide emission signalsthat may be detected by the light detectors.

In alternative embodiments, the biosensor may include electrodes orother types of sensors configured to detect other identifiableproperties. For example, the sensors may be configured to detect achange in ion concentration. In another example, the sensors may beconfigured to detect the ion current flow across a membrane

As used herein, a “cartridge” includes a structure that is configured tohold a biosensor. In some embodiments, the cartridge may includeadditional features, such as the light source (e.g., LEDs) that areconfigured to provide excitation light to the reactions sites of thebiosensor. The cartridge may also include a fluidic storage system(e.g., storage for reagents, sample, and buffer) and a fluidic controlsystem (e.g., pumps, valves, and the like) for fluidically transportingreaction components, sample, and the like to the reaction sites. Forexample, after the biosensor is prepared or manufactured, the biosensormay be coupled to a housing or container of the cartridge. In someembodiments, the biosensors and the cartridges may be self-contained,disposable units. However, other embodiments may include an assemblywith removable parts that allow a user to access an interior of thebiosensor or cartridge for maintenance or replacement of components orsamples. The biosensor and the cartridge may be removably coupled orengaged to larger bioassay systems, such as a sequencing system, thatconducts controlled reactions therein.

As used herein, when the terms “removably” and “coupled” (or “engaged”)are used together to describe a relationship between the biosensor (orcartridge) and a system receptacle or interface of a bioassay system,the term is intended to mean that a connection between the biosensor (orcartridge) and the system receptacle is readily separable withoutdestroying or damaging the system receptacle and/or the biosensor (orcartridge). Components are readily separable when the components may beseparated from each other without undue effort or a significant amountof time spent in separating the components. For example, the biosensor(or cartridge) may be removably coupled or engaged to the systemreceptacle in an electrical manner such that the mating contacts of thebioassay system are not destroyed or damaged. The biosensor (orcartridge) may also be removably coupled or engaged to the systemreceptacle in a mechanical manner such that the features that hold thebiosensor (or cartridge) are not destroyed or damaged. The biosensor (orcartridge) may also be removably coupled or engaged to the systemreceptacle in a fluidic manner such that the ports of the systemreceptacle are not destroyed or damaged. The system receptacle or acomponent is not considered to be destroyed or damaged if, for example,only a simple adjustment to the component (e.g., realignment) or asimple replacement (e.g., replacing a nozzle) is required.

As used herein, the term “fluid communication” or “fluidicly coupled”refers to two spatial regions being connected together such that aliquid or gas may flow between the two spatial regions. For example, amicrofluidic channel may be in fluid communication with a reactionchamber such that a fluid may flow freely into the reaction chamber fromthe microfluidic channel. The terms “in fluid communication” or“fluidicly coupled” allow for two spatial regions being in fluidcommunication through one or more valves, restrictors, or other fluidiccomponents that are configured to control or regulate a flow of fluidthrough a system.

As used herein, the term “immobilized,” when used with respect to abiomolecule or biological or chemical substance, includes substantiallyattaching the biomolecule or biological or chemical substance at amolecular level to a surface. For example, a biomolecule or biologicalor chemical substance may be immobilized to a surface of the substratematerial using adsorption techniques including non-covalent interactions(e.g., electrostatic forces, van der Waals, and dehydration ofhydrophobic interfaces) and covalent binding techniques where functionalgroups or linkers facilitate attaching the biomolecules to the surface.Immobilizing biomolecules or biological or chemical substances to asurface of a substrate material may be based upon the properties of thesubstrate surface, the liquid medium carrying the biomolecule orbiological or chemical substance, and the properties of the biomoleculesor biological or chemical substances themselves. In some cases, asubstrate surface may be functionalized (e.g., chemically or physicallymodified) to facilitate immobilizing the biomolecules (or biological orchemical substances) to the substrate surface. The substrate surface maybe first modified to have functional groups bound to the surface. Thefunctional groups may then bind to biomolecules or biological orchemical substances to immobilize them thereon. A substance can beimmobilized to a surface via a gel, for example, as described in USPatent Publ. No. US 2011/0059865 A1, which is incorporated herein byreference.

In some embodiments, nucleic acids can be attached to a surface andamplified using bridge amplification. Useful bridge amplificationmethods are described, for example, in U.S. Pat. No. 5,641,658; WO07/010251, U.S. Pat. No. 6,090,592; U.S. Patent Publ. No. 2002/0055100A1; U.S. Pat. No. 7,115,400; U.S. Patent Publ. No. 2004/0096853 A1; U.S.Patent Publ. No. 2004/0002090 A1; U.S. Patent Publ. No. 2007/0128624 A1;and U.S. Patent Publ. No. 2008/0009420 A1, each of which is incorporatedherein in its entirety. Another useful method for amplifying nucleicacids on a surface is rolling circle amplification (RCA), for example,using methods set forth in further detail below. In some embodiments,the nucleic acids can be attached to a surface and amplified using oneor more primer pairs. For example, one of the primers can be in solutionand the other primer can be immobilized on the surface (e.g.,5′-attached). By way of example, a nucleic acid molecule can hybridizeto one of the primers on the surface followed by extension of theimmobilized primer to produce a first copy of the nucleic acid. Theprimer in solution then hybridizes to the first copy of the nucleic acidwhich can be extended using the first copy of the nucleic acid as atemplate. Optionally, after the first copy of the nucleic acid isproduced, the original nucleic acid molecule can hybridize to a secondimmobilized primer on the surface and can be extended at the same timeor after the primer in solution is extended. In any embodiment, repeatedrounds of extension (e.g., amplification) using the immobilized primerand primer in solution provide multiple copies of the nucleic acid.

In particular embodiments, the assay protocols executed by the systemsand methods described herein include the use of natural nucleotides andalso enzymes that are configured to interact with the naturalnucleotides. Natural nucleotides include, for example, ribonucleotidesor deoxyribonucleotides. Natural nucleotides can be in the mono-, di-,or tri-phosphate form and can have a base selected from adenine (A),Thymine (T), uracil (U), guanine (G) or cytosine (C). It will beunderstood however that non-natural nucleotides, modified nucleotides oranalogs of the aforementioned nucleotides can be used. Some examples ofuseful non-natural nucleotides are set forth below in regard toreversible terminator-based sequencing by synthesis methods.

In embodiments that include reaction chambers, items or solid substances(including semi-solid substances) may be disposed within the reactionchambers. When disposed, the item or solid may be physically held orimmobilized within the reaction chamber through an interference fit,adhesion, or entrapment. Exemplary items or solids that may be disposedwithin the reaction chambers include polymer beads, pellets, agarosegel, powders, quantum dots, or other solids that may be compressedand/or held within the reaction chamber. In particular embodiments, anucleic acid superstructure, such as a DNA ball, can be disposed in orat a reaction chamber, for example, by attachment to an interior surfaceof the reaction chamber or by residence in a liquid within the reactionchamber. A DNA ball or other nucleic acid superstructure can bepreformed and then disposed in or at the reaction chamber.Alternatively, a DNA ball can be synthesized in situ at the reactionchamber. A DNA ball can be synthesized by rolling circle amplificationto produce a concatamer of a particular nucleic acid sequence and theconcatamer can be treated with conditions that form a relatively compactball. DNA balls and methods for their synthesis are described, forexample in, U.S. Patent Publ. Nos. 2008/0242560 A1 or 2008/0234136 A1,each of which is incorporated herein in its entirety. A substance thatis held or disposed in a reaction chamber can be in a solid, liquid, orgaseous state.

FIG. 1 is a block diagram of an exemplary bioassay system 100 forbiological or chemical analysis formed in accordance with oneembodiment. The term “bioassay” is not intended to be limiting as thebioassay system 100 may operate to obtain any information or data thatrelates to at least one of a biological or chemical substance. In someembodiments, the bioassay system 100 is a workstation that may besimilar to a bench-top device or desktop computer. For example, amajority (or all) of the systems and components for conducting thedesired reactions can be within a common housing 116.

In particular embodiments, the bioassay system 100 is a nucleic acidsequencing system (or sequencer) configured for various applications,including but not limited to de novo sequencing, resequencing of wholegenomes or target genomic regions, and metagenomics. The sequencer mayalso be used for DNA or RNA analysis. In some embodiments, the bioassaysystem 100 may also be configured to generate reaction sites in abiosensor. For example, the bioassay system 100 may be configured toreceive a sample and generate surface attached clusters of clonallyamplified nucleic acids derived from the sample. Each cluster mayconstitute or be part of a reaction site in the biosensor.

The exemplary bioassay system 100 may include a system receptacle orinterface 112 that is configured to interact with a biosensor 102 toperform desired reactions within the biosensor 102. In the followingdescription with respect to FIG. 1, the biosensor 102 is loaded into thesystem receptacle 112. However, it is understood that a cartridge thatincludes the biosensor 102 may be inserted into the system receptacle112 and in some states the cartridge can be removed temporarily orpermanently. As described above, the cartridge may include, among otherthings, fluidic control and fluidic storage components.

In particular embodiments, the bioassay system 100 is configured toperform a large number of parallel reactions within the biosensor 102.The biosensor 102 includes one or more reaction sites where desiredreactions can occur. The reaction sites may be, for example, immobilizedto a solid surface of the biosensor or immobilized to beads (or othermovable substrates) that are located within corresponding reactionchambers of the biosensor. The reaction sites can include, for example,clusters of clonally amplified nucleic acids. The biosensor 102 mayinclude a solid-state imaging device (e.g., CCD or CMOS imager) and aflow cell mounted thereto. The flow cell may include one or more flowchannels that receive a solution from the bioassay system 100 and directthe solution toward the reaction sites. Optionally, the biosensor 102can be configured to engage a thermal element for transferring thermalenergy into or out of the flow channel.

The bioassay system 100 may include various components, assemblies, andsystems (or sub-systems) that interact with each other to perform apredetermined method or assay protocol for biological or chemicalanalysis. For example, the bioassay system 100 includes a systemcontroller 104 that may communicate with the various components,assemblies, and sub-systems of the bioassay system 100 and also thebiosensor 102. For example, in addition to the system receptacle 112,the bioassay system 100 may also include a fluidic control system 106 tocontrol the flow of fluid throughout a fluid network of the bioassaysystem 100 and the biosensor 102; a fluid storage system 108 that isconfigured to hold all fluids (e.g., gas or liquids) that may be used bythe bioassay system; a temperature control system 110 that may regulatethe temperature of the fluid in the fluid network, the fluid storagesystem 108, and/or the biosensor 102; and an illumination system 111that is configured to illuminate the biosensor 102. As described above,if a cartridge having the biosensor 102 is loaded into the systemreceptacle 112, the cartridge may also include fluidic control andfluidic storage components.

Also shown, the bioassay system 100 may include a user interface 114that interacts with the user. For example, the user interface 114 mayinclude a display 113 to display or request information from a user anda user input device 115 to receive user inputs. In some embodiments, thedisplay 113 and the user input device 115 are the same device. Forexample, the user interface 114 may include a touch-sensitive displayconfigured to detect the presence of an individual's touch and alsoidentify a location of the touch on the display. However, other userinput devices 115 may be used, such as a mouse, touchpad, keyboard,keypad, handheld scanner, voice-recognition system, motion-recognitionsystem, and the like. As will be discussed in greater detail below, thebioassay system 100 may communicate with various components, includingthe biosensor 102 (e.g. in the form of a cartridge), to perform thedesired reactions. The bioassay system 100 may also be configured toanalyze data obtained from the biosensor to provide a user with desiredinformation.

The system controller 104 may include any processor-based ormicroprocessor-based system, including systems using microcontrollers,reduced instruction set computers (RISC), application specificintegrated circuits (ASICs), field programmable gate array (FPGAs),logic circuits, and any other circuit or processor capable of executingfunctions described herein. The above examples are exemplary only, andare thus not intended to limit in any way the definition and/or meaningof the term system controller. In the exemplary embodiment, the systemcontroller 104 executes a set of instructions that are stored in one ormore storage elements, memories, or modules in order to at least one ofobtain and analyze detection data. Storage elements may be in the formof information sources or physical memory elements within the bioassaysystem 100.

The set of instructions may include various commands that instruct thebioassay system 100 or biosensor 102 to perform specific operations suchas the methods and processes of the various embodiments describedherein. The set of instructions may be in the form of a softwareprogram, which may form part of a tangible, non-transitory computerreadable medium or media. As used herein, the terms “software” and“firmware” are interchangeable, and include any computer program storedin memory for execution by a computer, including RAM memory, ROM memory,EPROM memory, EEPROM memory, and non-volatile RAM (NVRAM) memory. Theabove memory types are exemplary only, and are thus not limiting as tothe types of memory usable for storage of a computer program.

The software may be in various forms such as system software orapplication software. Further, the software may be in the form of acollection of separate programs, or a program module within a largerprogram or a portion of a program module. The software also may includemodular programming in the form of object-oriented programming. Afterobtaining the detection data, the detection data may be automaticallyprocessed by the bioassay system 100, processed in response to userinputs, or processed in response to a request made by another processingmachine (e.g., a remote request through a communication link).

The system controller 104 may be connected to the biosensor 102 and theother components of the bioassay system 100 via communication links. Thesystem controller 104 may also be communicatively connected to off-sitesystems or servers. The communication links may be hardwired orwireless. The system controller 104 may receive user inputs or commands,from the user interface 114 and the user input device 115.

The fluidic control system 106 includes a fluid network and isconfigured to direct and regulate the flow of one or more fluids throughthe fluid network. The fluid network may be in fluid communication withthe biosensor 102 and the fluid storage system 108. For example, selectfluids may be drawn from the fluid storage system 108 and directed tothe biosensor 102 in a controlled manner, or the fluids may be drawnfrom the biosensor 102 and directed toward, for example, a wastereservoir in the fluid storage system 108. Although not shown, thefluidic control system 106 may include flow sensors that detect a flowrate or pressure of the fluids within the fluid network. The sensors maycommunicate with the system controller 104.

The temperature control system 110 is configured to regulate thetemperature of fluids at different regions of the fluid network, thefluid storage system 108, and/or the biosensor 102. For example, thetemperature control system 110 may include a thermocycler thatinterfaces with the biosensor 102 and controls the temperature of thefluid that flows along the reaction sites in the biosensor 102. Thetemperature control system 110 may also regulate the temperature ofsolid elements or components of the bioassay system 100 or the biosensor102. Although not shown, the temperature control system 110 may includesensors to detect the temperature of the fluid or other components. Thesensors may communicate with the system controller 104.

The fluid storage system 108 is in fluid communication with thebiosensor 102 and may store various reaction components or reactantsthat are used to conduct the desired reactions therein. The fluidstorage system 108 may also store fluids for washing or cleaning thefluid network and biosensor 102 and for diluting the reactants. Forexample, the fluid storage system 108 may include various reservoirs tostore samples, reagents, enzymes, other biomolecules, buffer solutions,aqueous, and non-polar solutions, and the like. Furthermore, the fluidstorage system 108 may also include waste reservoirs for receiving wasteproducts from the biosensor 102. In embodiments that include acartridge, the cartridge may include one or more of a fluid storagesystem, fluidic control system or temperature control system.Accordingly, one or more of the components set forth herein as relatingto those systems can be contained within a cartridge housing. Forexample, a cartridge can have various reservoirs to store samples,reagents, enzymes, other biomolecules, buffer solutions, aqueous, andnon-polar solutions, waste, and the like. As such, one or more of afluid storage system, fluidic control system or temperature controlsystem can be removably engaged with a bioassay system via a cartridgeor other biosensor.

The illumination system 111 may include a light source (e.g., one ormore LEDs) and a plurality of optical components to illuminate thebiosensor. Examples of light sources may include lasers, arc lamps,LEDs, or laser diodes. The optical components may be, for example,reflectors, dichroics, beam splitters, collimators, lenses, filters,wedges, prisms, mirrors, detectors, and the like. In embodiments thatuse an illumination system, the illumination system 111 may beconfigured to direct an excitation light to reaction sites. As oneexample, fluorophores may be excited by green wavelengths of light, assuch the wavelength of the excitation light may be approximately 532 nm.

The system receptacle or interface 112 is configured to engage thebiosensor 102 in at least one of a mechanical, electrical, and fluidicmanner. The system receptacle 112 may hold the biosensor 102 in adesired orientation to facilitate the flow of fluid through thebiosensor 102. The system receptacle 112 may also include electricalcontacts that are configured to engage the biosensor 102 so that thebioassay system 100 may communicate with the biosensor 102 and/orprovide power to the biosensor 102. Furthermore, the system receptacle112 may include fluidic ports (e.g., nozzles) that are configured toengage the biosensor 102. In some embodiments, the biosensor 102 isremovably coupled to the system receptacle 112 in a mechanical manner,in an electrical manner, and also in a fluidic manner.

In addition, the bioassay system 100 may communicate remotely with othersystems or networks or with other bioassay systems 100. Detection dataobtained by the bioassay system(s) 100 may be stored in a remotedatabase.

FIG. 2 is a block diagram of the system controller 104 in the exemplaryembodiment. In one embodiment, the system controller 104 includes one ormore processors or modules that can communicate with one another. Eachof the processors or modules may include an algorithm (e.g.,instructions stored on a tangible and/or non-transitory computerreadable storage medium) or sub-algorithms to perform particularprocesses. The system controller 104 is illustrated conceptually as acollection of modules, but may be implemented utilizing any combinationof dedicated hardware boards, DSPs, processors, etc. Alternatively, thesystem controller 104 may be implemented utilizing an off-the-shelf PCwith a single processor or multiple processors, with the functionaloperations distributed between the processors. As a further option, themodules described below may be implemented utilizing a hybridconfiguration in which certain modular functions are performed utilizingdedicated hardware, while the remaining modular functions are performedutilizing an off-the-shelf PC and the like. The modules also may beimplemented as software modules within a processing unit.

During operation, a communication link 120 may transmit information(e.g. commands) to or receive information (e.g. data) from the biosensor102 (FIG. 1) and/or the sub-systems 106, 108, 110 (FIG. 1). Acommunication link 122 may receive user input from the user interface114 (FIG. 1) and transmit data or information to the user interface 114.Data from the biosensor 102 or sub-systems 106, 108, 110 may beprocessed by the system controller 104 in real-time during a bioassaysession. Additionally or alternatively, data may be stored temporarilyin a system memory during a bioassay session and processed in slowerthan real-time or off-line operation.

As shown in FIG. 2, the system controller 104 may include a plurality ofmodules 131-139 that communicate with a main control module 130. Themain control module 130 may communicate with the user interface 114(FIG. 1). Although the modules 131-139 are shown as communicatingdirectly with the main control module 130, the modules 131-139 may alsocommunicate directly with each other, the user interface 114, and thebiosensor 102. Also, the modules 131-139 may communicate with the maincontrol module 130 through the other modules.

The plurality of modules 131-139 include system modules 131-133, 139that communicate with the sub-systems 106, 108, 110, and 111,respectively. The fluidic control module 131 may communicate with thefluidic control system 106 to control the valves and flow sensors of thefluid network for controlling the flow of one or more fluids through thefluid network. The fluid storage module 132 may notify the user whenfluids are low or when the waste reservoir is at or near capacity. Thefluid storage module 132 may also communicate with the temperaturecontrol module 133 so that the fluids may be stored at a desiredtemperature. The illumination module 139 may communicate with theillumination system 109 to illuminate the reaction sites at designatedtimes during a protocol, such as after the desired reactions (e.g.,binding events) have occurred.

The plurality of modules 131-139 may also include a device module 134that communicates with the biosensor 102 and an identification module135 that determines identification information relating to the biosensor102. The device module 134 may, for example, communicate with the systemreceptacle 112 to confirm that the biosensor has established anelectrical and fluidic connection with the bioassay system 100. Theidentification module 135 may receive signals that identify thebiosensor 102. The identification module 135 may use the identity of thebiosensor 102 to provide other information to the user. For example, theidentification module 135 may determine and then display a lot number, adate of manufacture, or a protocol that is recommended to be run withthe biosensor 102.

The plurality of modules 131-139 may also include a detection dataanalysis module 138 that receives and analyzes the signal data (e.g.,image data) from the biosensor 102. The signal data may be stored forsubsequent analysis or may be transmitted to the user interface 114 todisplay desired information to the user. In some embodiments, the signaldata may be processed by the solid-state imager (e.g., CMOS imagesensor) before the detection data analysis module 138 receives thesignal data.

Protocol modules 136 and 137 communicate with the main control module130 to control the operation of the sub-systems 106, 108, and 110 whenconducting predetermined assay protocols. The protocol modules 136 and137 may include sets of instructions for instructing the bioassay system100 to perform specific operations pursuant to predetermined protocols.As shown, the protocol module may be a sequencing-by-synthesis (SBS)module 136 that is configured to issue various commands for performingsequencing-by-synthesis processes. In SBS, extension of a nucleic acidprimer along a nucleic acid template is monitored to determine thesequence of nucleotides in the template. The underlying chemical processcan be polymerization (e.g. as catalyzed by a polymerase enzyme) orligation (e.g. catalyzed by a ligase enzyme). In a particularpolymerase-based SBS embodiment, fluorescently labeled nucleotides areadded to a primer (thereby extending the primer) in a template dependentfashion such that detection of the order and type of nucleotides addedto the primer can be used to determine the sequence of the template. Forexample, to initiate a first SBS cycle, commands can be given to deliverone or more labeled nucleotides, DNA polymerase, etc., into/through aflow cell that houses an array of nucleic acid templates. The nucleicacid templates may be located at corresponding reaction sites. Thosereaction sites where primer extension causes a labeled nucleotide to beincorporated can be detected through an imaging event. During an imagingevent, the illumination system 111 may provide an excitation light tothe reaction sites. Optionally, the nucleotides can further include areversible termination property that terminates further primer extensiononce a nucleotide has been added to a primer. For example, a nucleotideanalog having a reversible terminator moiety can be added to a primersuch that subsequent extension cannot occur until a deblocking agent isdelivered to remove the moiety. Thus, for embodiments that usereversible termination a command can be given to deliver a deblockingreagent to the flow cell (before or after detection occurs). One or morecommands can be given to effect wash(es) between the various deliverysteps. The cycle can then be repeated n times to extend the primer by nnucleotides, thereby detecting a sequence of length n. Exemplarysequencing techniques are described, for example, in Bentley et al.,Nature 456:53-59 (2008), WO 04/018497; U.S. Pat. No. 7,057,026; WO91/06678; WO 07/123744; U.S. Pat. Nos. 7,329,492; 7,211,414; 7,315,019;7,405,281, and US 2008/0108082, each of which is incorporated herein byreference.

For the nucleotide delivery step of an SBS cycle, either a single typeof nucleotide can be delivered at a time, or multiple differentnucleotide types (e.g. A, C, T and G together) can be delivered. For anucleotide delivery configuration where only a single type of nucleotideis present at a time, the different nucleotides need not have distinctlabels since they can be distinguished based on temporal separationinherent in the individualized delivery. Accordingly, a sequencingmethod or apparatus can use single color detection. For example, anexcitation source need only provide excitation at a single wavelength orin a single range of wavelengths. For a nucleotide deliveryconfiguration where delivery results in multiple different nucleotidesbeing present in the flow cell at one time, sites that incorporatedifferent nucleotide types can be distinguished based on differentfluorescent labels that are attached to respective nucleotide types inthe mixture. For example, four different nucleotides can be used, eachhaving one of four different fluorophores. In one embodiment, the fourdifferent fluorophores can be distinguished using excitation in fourdifferent regions of the spectrum. For example, four differentexcitation radiation sources can be used. Alternatively, fewer than fourdifferent excitation sources can be used, but optical filtration of theexcitation radiation from a single source can be used to producedifferent ranges of excitation radiation at the flow cell.

In some embodiments, fewer than four different colors can be detected ina mixture having four different nucleotides. For example, pairs ofnucleotides can be detected at the same wavelength, but distinguishedbased on a difference in intensity for one member of the pair comparedto the other, or based on a change to one member of the pair (e.g. viachemical modification, photochemical modification or physicalmodification) that causes apparent signal to appear or disappearcompared to the signal detected for the other member of the pair.Exemplary apparatus and methods for distinguishing four differentnucleotides using detection of fewer than four colors are described forexample in U.S. Pat. App. Ser. Nos. 61/538,294 and 61/619,878, which areincorporated herein by reference their entireties. U.S. application Ser.No. 13/624,200, which was filed on Sep. 21, 2012, is also incorporatedby reference in its entirety.

The plurality of protocol modules may also include a sample-preparation(or generation) module 137 that is configured to issue commands to thefluidic control system 106 and the temperature control system 110 foramplifying a product within the biosensor 102. For example, thebiosensor 102 may be engaged to the bioassay system 100. Theamplification module 137 may issue instructions to the fluidic controlsystem 106 to deliver necessary amplification components to reactionchambers within the biosensor 102. In other embodiments, the reactionsites may already contain some components for amplification, such as thetemplate DNA and/or primers. After delivering the amplificationcomponents to the reaction chambers, the amplification module 137 mayinstruct the temperature control system 110 to cycle through differenttemperature stages according to known amplification protocols. In someembodiments, the amplification and/or nucleotide incorporation isperformed isothermally.

The SBS module 136 may issue commands to perform bridge PCR whereclusters of clonal amplicons are formed on localized areas within achannel of a flow cell. After generating the amplicons through bridgePCR, the amplicons may be “linearized” to make single stranded templateDNA, or sstDNA, and a sequencing primer may be hybridized to a universalsequence that flanks a region of interest. For example, a reversibleterminator-based sequencing by synthesis method can be used as set forthabove or as follows.

Each sequencing cycle can extend a sstDNA by a single base which can beaccomplished for example by using a modified DNA polymerase and amixture of four types of nucleotides. The different types of nucleotidescan have unique fluorescent labels, and each nucleotide can further havea reversible terminator that allows only a single-base incorporation tooccur in each cycle. After a single base is added to the sstDNA,excitation light may be incident upon the reaction sites and fluorescentemissions may be detected. After detection, the fluorescent label andthe terminator may be chemically cleaved from the sstDNA. Anothersimilar sequencing cycle may follow. In such a sequencing protocol, theSBS module 136 may instruct the fluidic control system 106 to direct aflow of reagent and enzyme solutions through the biosensor 102.Exemplary reversible terminator-based SBS methods which can be utilizedwith the apparatus and methods set forth herein are described in USPatent Application Publication No. 2007/0166705 A1, US PatentApplication Publication No. 2006/0188901 A1, U.S. Pat. No. 7,057,026, USPatent Application Publication No. 2006/0240439 A1, US PatentApplication Publication No. 2006/0281109 A1, PCT Publication No. WO05/065814, US Patent Application Publication No. 2005/0100900 A1, PCTPublication No. WO 06/064199 and PCT Publication No. WO 07/010251, eachof which is incorporated herein by reference in its entirety. Exemplaryreagents for reversible terminator-based SBS are described in U.S. Pat.Nos. 7,541,444; 7,057,026; 7,414,116; 7,427,673; 7,566,537; 7,592,435and WO 07/135368, each of which is incorporated herein by reference inits entirety.

In some embodiments, the amplification and SBS modules may operate in asingle assay protocol where, for example, template nucleic acid isamplified and subsequently sequenced within the same cartridge.

The bioassay system 100 may also allow the user to reconfigure an assayprotocol. For example, the bioassay system 100 may offer options to theuser through the user interface 114 for modifying the determinedprotocol. For example, if it is determined that the biosensor 102 is tobe used for amplification, the bioassay system 100 may request atemperature for the annealing cycle. Furthermore, the bioassay system100 may issue warnings to a user if a user has provided user inputs thatare generally not acceptable for the selected assay protocol.

FIG. 3 is a block diagram of an exemplary workstation 200 for biologicalor chemical analysis in accordance with one embodiment. The workstation200 may have similar features, systems, and assemblies as the bioassaysystem 100 described above. For example, the workstation 200 may have afluidic control system, such as the fluidic control system 106 (FIG. 1),that is fluidicly coupled to a biosensor (or cartridge) 235 through afluid network 238. The fluid network 238 may include a reagent cartridge240, a valve block 242, a main pump 244, a debubbler 246, a 3-way valve248, a flow restrictor 250, a waste removal system 252, and a purge pump254. In particular embodiments, most of the components or all of thecomponents described above are within a common workstation housing (notshown). Although not shown, the workstation 200 may also include anillumination system, such as the illumination system 111, that isconfigured to provide an excitation light to the reaction sites.

A flow of fluid is indicated by arrows along the fluid network 238. Forexample, reagent solutions may be removed from the reagent cartridge 240and flow through the valve block 242. The valve block 242 may facilitatecreating a zero-dead volume of the fluid flowing to the cartridge 235from the reagent cartridge 240. The valve block 242 can select or permitone or more liquids within the reagent cartridge 240 to flow through thefluid network 238. For example, the valve block 242 can include solenoidvalves that have a compact arrangement. Each solenoid valve can controlthe flow of a fluid from a single reservoir bag. In some embodiments,the valve block 242 can permit two or more different liquids to flowinto the fluid network 238 at the same time thereby mixing the two ormore different liquids. After leaving the valve block 242, the fluid mayflow through the main pump 244 and to the debubbler 246. The debubbler246 is configured to remove unwanted gases that have entered or beengenerated within the fluid network 238.

From the debubbler 246, fluid may flow to the 3-way valve 248 where thefluid is either directed to the cartridge 235 or bypassed to the wasteremoval system 252. A flow of the fluid within the cartridge 235 may beat least partially controlled by the flow restrictor 250 locateddownstream from the cartridge 235. Furthermore, the flow restrictor 250and the main pump 244 may coordinate with each other to control the flowof fluid across reaction sites and/or control the pressure within thefluid network 238. Fluid may flow through the cartridge 235 and onto thewaste removal system 252. Optionally, fluid may flow through the purgepump 254 and into, for example, a waste reservoir bag within the reagentcartridge 240.

Also shown in FIG. 3, the workstation 200 may include a temperaturecontrol system, such as the temperature control system 110, that isconfigured to regulate or control a thermal environment of the differentcomponents and sub-systems of the workstation 200. The temperaturecontrol system 110 can include a reagent cooler 264 that is configuredto control the temperature requirements of various fluids used by theworkstation 200, and a thermocycler 266 that is configured to controlthe temperature of a cartridge 235. The thermocycler 266 can include athermal element (not shown) that interfaces with the cartridge.

Furthermore, the workstation 200 may include a system controller or SBSboard 260 that may have similar features as the system controller 104described above. The SBS board 260 may communicate with the variouscomponents and sub-systems of the workstation 200 as well as thecartridge 235. Furthermore, the SBS board 260 may communicate withremote systems to, for example, store data or receive commands from theremote systems. The workstation 200 may also include a touch screen userinterface 262 that is operatively coupled to the SBS board 260 through asingle-board computer (SBC) 272. The workstation 200 may also includeone or more user accessible data communication ports and/or drives. Forexample a workstation 200 may include one or more universal serial bus(USB) connections for computer peripherals, such as a flash or jumpdrive, a compact-flash (CF) drive and/or a hard drive 270 for storinguser data in addition to other software.

FIG. 4 is a perspective view of a workstation 300 and a cartridge 302that may include one or more biosensors (not shown) as described herein.The workstation 300 may include similar components as described abovewith respect to the bioassay system 100 and the workstation 200 and mayoperate in a similar manner. For example, the workstation 300 mayinclude a workstation housing 304 and a system receptacle 306 that isconfigured to receive and engage the cartridge 302. The systemreceptacle may at least one of fluidically or electrically engage thecartridge 302. The workstation housing 304 may hold, for example, asystem controller, a fluid storage system, a fluidic control system, anda temperature control system as described above. In FIG. 4, theworkstation 300 does not include a user interface or display that iscoupled to the workstation housing 304. However, a user interface may becommunicatively coupled to the housing 304 (and the components/systemstherein) through a communication link. Thus, the user interface and theworkstation 300 may be remotely located with respect to each other.Together, the user interface and the workstation 300 (or a plurality ofworkstations) may constitute a bioassay system.

As shown, the cartridge 302 includes a cartridge housing 308 having atleast one port 310 that provides access to an interior of the cartridgehousing 308. For example, a solution that is configured to be used inthe cartridge 302 during the controlled reactions may be insertedthrough the port 310 by a technician or by the workstation 300. Thesystem receptacle 306 and the cartridge 302 may be sized and shapedrelative to each other such that the cartridge 302 may be inserted intoa receptacle cavity (not shown) of the system receptacle 306.

FIG. 5 is a front view of a rack assembly 312 having a cabinet orcarriage 314 with a plurality of the workstations 300 loaded thereon.The cabinet 314 may include one or more shelves 316 that define one ormore reception spaces 318 configured to receive one or more workstations300. Although not shown, the workstations 300 may be communicativelycoupled to a communication network that permits a user to controloperation of the workstations 300. In some embodiments, a bioassaysystem includes a plurality of workstations, such as the workstations300, and a single user interface configured to control operation of themultiple workstations.

FIG. 6 illustrates various features of the cartridge 302 (FIG. 4) inaccordance with one embodiment. As shown, the cartridge 302 may includea sample assembly 320, and the system receptacle 306 may include a lightassembly 322. Stage 346 shown in FIG. 6 represents the spatialrelationship between the first and second sub-assemblies 320 and 322when they are separate from each other. At stage 348, the first andsecond sub-assemblies 320 and 322 are joined together. The cartridgehousing 308 (FIG. 4) may enclose the joined first and secondsub-assemblies 320 and 322.

In the illustrated embodiment, the first sub-assembly 320 includes abase 326 and a reaction-component body 324 that is mounted onto the base326. Although not shown, one or more biosensors may be mounted to thebase 326 in a recess 328 that is defined, at least in part, by thereaction-component body 324 and the base 326. For example, at least fourbiosensors may be mounted to the base 326. In some embodiments, the base326 is a printed circuit board having circuitry that enablescommunication between the different components of the cartridge and theworkstation 300 (FIG. 4). For example, the reaction-component body 324may include a rotary valve 330 and reagent reservoirs 332 that arefluidically coupled to the rotary valve 330. The reaction-component body324 may also include additional reservoirs 334.

The second sub-assembly 322 includes a light assembly 336 that includesa plurality of light directing channels 338. Each light directingchannel 338 is optically coupled to a light source (not shown), such asa light-emitting diode (LED). The light source(s) are configured toprovide an excitation light that is directed by the light directingchannels 338 onto the biosensors. In alternative embodiments, thecartridge may not include a light source(s). In such embodiments, thelight source(s) may be located in the workstation 300. When thecartridge is inserted into the system receptacle 306 (FIG. 4), thecartridge 302 may align with the light source(s) so that the biosensorsmay be illuminated.

Also shown in FIG. 6, the second sub-assembly 322 includes a cartridgepump 340 that is fluidically coupled to ports 342 and 344. When thefirst and second sub-assemblies 320 and 322 are joined together, theport 342 is coupled to the rotary valve 330 and the port 344 is coupledto the other reservoirs 334. The cartridge pump 340 may be activated todirect reaction components from the reservoirs 332 and/or 334 to thebiosensors according to a designated protocol.

FIG. 7 illustrates a cross-section of an exemplary biosensor 400 formedin accordance with one embodiment. The biosensor 400 may have similarfeatures as the biosensor 102 (FIG. 1) described above and may be usedin, for example, the cartridge 302 (FIG. 4). As shown, the biosensor 400may include a flow cell 402 that is mounted onto a detection device 404.In the illustrated embodiment, the flow cell 402 is affixed directly tothe detection device 404. However, in alternative embodiments, the flowcell 402 may be removably coupled to the detection device 404. Thedetection device 404 has a detector surface 412 that may befunctionalized (e.g., chemically or physically modified in a suitablemanner for conducting the desired reactions). For example, the detectorsurface 412 may be functionalized and may include a plurality ofreaction sites 414 having one or more biomolecules immobilized thereto.In the illustrated embodiment, the flow cell 402 includes sidewalls 406,408 and a flow cover 410 that is supported by the sidewalls 406, 408.The sidewalls 406, 408 are coupled to the detector surface 412 andextend between the flow cover 410 and the sidewalls 406, 408. In someembodiments, the sidewalls 406, 408 are formed from a curable adhesivelayer that bonds the flow cover 410 to the detection device 404.

The sidewalls 406, 408 are sized and shaped so that a flow channel 418exists between the flow cover 410 and the detection device 404. Asshown, the flow channel 418 may include a height H₁ that is determinedby the sidewalls 406, 408. The height H₁ may be between about 50-400 μm(microns) or, more particularly, about 80-200 μm. In the illustratedembodiment, the height H₁ is about 100 μm. The flow cover 410 mayinclude a material that is transparent to excitation light 401propagating from an exterior of the biosensor 400 into the flow channel418. As shown in FIG. 7, the excitation light 401 approaches the flowcover 410 at a non-orthogonal angle. However, this is only forillustrative purposes as the excitation light 401 may approach the flowcover 410 from different angles.

Also shown, the flow cover 410 may include inlet and outlet ports 420,422 that are configured to fluidically engage other ports (not shown).For example, the other ports may be from the cartridge 302 (FIG. 4) orthe workstation 300 (FIG. 4). The flow channel 418 is sized and shapedto direct a fluid along the detector surface 412. The height H₁ andother dimensions of the flow channel 418 may be configured to maintain asubstantially even flow of a fluid along the detector surface 412. Thedimensions of the flow channel 418 may also be configured to controlbubble formation.

As shown in exemplary FIG. 7, the sidewalls 406, 408 and the flow cover410 are separate components that are coupled to each other. Inalternative embodiments, the sidewalls 406, 408 and the flow cover 410may be integrally formed such that the sidewalls 406, 408 and the flowcover 410 are formed from a continuous piece of material. By way ofexample, the flow cover 410 (or the flow cell 402) may comprise atransparent material, such as glass or plastic. The flow cover 410 mayconstitute a substantially rectangular block having a planar exteriorsurface and a planar inner surface that defines the flow channel 418.The block may be mounted onto the sidewalls 406, 408. Alternatively, theflow cell 402 may be etched to define the flow cover 410 and thesidewalls 406, 408. For example, a recess may be etched into thetransparent material. When the etched material is mounted to thedetection device 404, the recess may become the flow channel 418.

The detector surface 412 may be substantially planar as shown in FIG. 7.However, in alternative embodiments, the detector surface 412 may beshaped to define reaction chambers in which each reaction chamber hasone or more reaction sites 414. The reaction chambers may be defined by,for example, chamber walls that effectively separate the reactionsite(s) 414 of one reaction chamber from the reaction site(s) 414 of anadjacent reaction chamber.

As shown in FIG. 7, the reaction sites 414 may be distributed in apattern along the detector surface 412. For example, the reactions sites414 may be located in rows and columns along the detector surface 412 ina manner that is similar to a microarray. However, it is understood thatvarious patterns of reaction sites may be used. In particularembodiments, the reaction sites 414 include clusters or colonies ofbiomolecules (e.g., oligonucleotides) that are immobilized on thedetector surface 412.

The detection device 404 may be similar to, for example, an integratedcircuit comprising a plurality of stacked substrate layers 431-434. Thesubstrate layers 431-434 may include a base substrate 431, a solid-stateimager 432 (e.g., CMOS image sensor), a filter or light-management layer433, and a passivation layer 434. It should be noted that the above isonly illustrative and that other embodiments may include fewer oradditional layers. Moreover, each of the substrate layers 431-434 mayinclude a plurality of sub-layers. As will be described in greaterdetail below, the detection device 404 may be manufactured usingprocesses that are similar to those used in manufacturing integratedcircuits, such as CMOS image sensors and CCDs. For example, thesubstrate layers 431-434 or portions thereof may be grown, deposited,etched, and the like to form the detection device 404.

The passivation layer 434 is configured to shield the filter layer 433from the fluidic environment of the flow channel 418. In some cases, thepassivation layer 434 is also configured to provide a solid surface(i.e., the detector surface 412) that permits biomolecules or otheranalytes-of-interest to be immobilized thereon. For example, each of thereaction sites 414 may include a cluster of biomolecules that areimmobilized to the detector surface 412. Thus, the passivation layer 434may be formed from a material that permits the reaction sites 414 to beimmobilized thereto. The passivation layer 434 may also comprise amaterial that is at least transparent to a desired fluorescent light. Byway of example, the passivation layer 434 may include silicon nitride(Si₃N₄) and/or silica (Sift). However, other suitable material(s) may beused. In the illustrated embodiment, the passivation layer 434 may besubstantially planar. However, in alternative embodiments, thepassivation layer 434 may include recesses, such as pits, wells,grooves, and the like. In the illustrated embodiment, the passivationlayer 434 has a thickness that is about 150-200 nm and, moreparticularly, about 170 nm.

The filter layer 433 may include various features that affect thetransmission of light. In some embodiments, the filter layer 433 canperform multiple functions. For instance, the filter layer 433 may beconfigured to (a) filter unwanted light signals, such as light signalsfrom an excitation light source; (b) direct emission signals from thereaction sites 414 toward corresponding light detectors 436 that areconfigured to detect the emission signals from the reaction sites; or(c) block or prevent detection of unwanted emission signals fromadjacent reaction sites. As such, the filter layer 433 may also bereferred to as a light-management layer. In the illustrated embodiment,the filter layer 433 has a thickness that is about 1-5 μm and, moreparticularly, about 3-4 μm.

In some embodiments, the filter layer 433 may include a plurality offilter walls 440. The filter walls 440 may be configured to at least oneof (a) reflect emission signals or (b) block or prevent unwantedemissions signals from adjacent reaction sites. As will be described ingreater detail below, adjacent filter walls 440, such as filter walls440A, 440B, may define a detection path 445 for the emission signalsthat are provided by the reaction site 414′. The detection path 445extends between the detector surface 412 to a light detector 436, whichis described in greater detail below. The filter walls 440 may be formedfrom various kinds of materials. For example, the filter walls 440 mayinclude an internal material 442 (e.g., glass) and an exterior coating444 that is deposited onto surfaces of the internal material 442. In theillustrated embodiment, the coating 444 includes a reflective metal(e.g., aluminum). In alternative embodiments, the coating 444 mayinclude a dielectric material.

Also shown, a light-absorbing material 446 may be deposited betweenadjacent filter walls 440. The light-absorbing material 446 may include,for example, a material that is configured to absorb the excitationlight and permit the fluorescent emissions (i.e., emission light,emission signals) to pass therethrough. In the illustrated embodiment,the light-absorbing material 446 may comprise a resist-based absorptionmaterial that is configured to block, for example, 532 nm excitationlight. However, other light-absorbing materials 446 may be used. Inalternative embodiments, a dichroic filter may be positioned betweenadjacent filter walls 440. In other alternative embodiments, a dichroicfilter layer is located above or below the filter walls 440. Forexample, the dichroic filter layer may be located between thepassivation layer 434 and the filter layer 433.

In alternative embodiments, the filter layer 433 may include an array ofmicrolenses or other optical components. Each of the microlenses may beconfigured to direct emission signals from an associated reaction site414 to an associated light detector 436. Such microlenses may be used inaddition to or as an alternative to the filter walls 440.

In some embodiments, the solid-state imager 432 and the base substrate431 may be provided together as a previously constructed solid-stateimaging device (e.g., CMOS chip). For example, the base substrate 431may be a wafer of silicon and the solid-state imager 432 may be mountedthereon. The solid-state imager 432 includes a layer of semiconductormaterial (e.g., silicon) and the light detectors 436. In someembodiments, each light detector 436 is formed from a single pixel. Inother embodiments, multiple pixels (e.g., 2, 3, 4, 5, 6, or more) mayform a single light detector 436. In the illustrated embodiment, thelight detector 436 pixels are photodiodes configured to detect light.

The solid-state imager 432 may include a dense array of light detectors436 that are configured to detect activity indicative of a desiredreaction from within or along the flow channel 418. In some embodiments,each light detector 436 has a detection area that is less than about 50μm². In particular embodiments, the detection area is less than about 10μm². In more particular embodiments, the detection area is about 2 μm².In such cases, the light detector 436 may constitute a single pixel. Anaverage read noise of each pixel in a light detector 436 may be, forexample, less than about 150 electrons. In more particular embodiments,the read noise may be less than about 5 electrons. The resolution of thearray of light detectors 436 may be greater than about 0.5 megapixels(Mpixels). In more specific embodiments, the resolution may be greaterthan about 5 Mpixels and, more particularly, greater than about 10Mpixels.

In some embodiments, the detection device 404 includes a microcircuitarrangement, such as the microcircuit arrangement described in U.S. Pat.No. 7,595,883, which is incorporated herein by reference in theentirety. More specifically, the detection device 404 may comprise anintegrated circuit having a planar array of the light detectors 436.Circuitry formed within the detection device 404 may be configured forat least one of signal amplification, digitization, storage, andprocessing. The circuitry may collect and analyze the detectedfluorescent light and generate data signals for communicating detectiondata to a bioassay system. The circuitry may also perform additionalanalog and/or digital signal processing in the detection device 404. Thecircuitry may include conductive vias 450 that transmit the data signalsto a bottom side 452 of the biosensor 400. The data signals may betransmitted through electrical contacts 454 of the biosensor 400. Bytransmitting the data signals to the bottom side 452 instead of a topside 453, the biosensor 400 may be permitted to use more area of the topside 453 for detecting fluorescent light.

FIG. 8 is a flowchart illustrating a method or workflow 500 ofmanufacturing the biosensor 400 shown in FIG. 7. However, it should benoted that the method 500 is only one example of manufacturing thebiosensor 400 and others may be used. FIGS. 9-15 illustrate thebiosensor 400 at different manufacturing stages throughout the workflow.The method 500 includes providing at 502 the solid-state imager 432. Theproviding operation 502 may include providing the solid-state imager 432with the base substrate 431 attached thereto. For example, in someembodiments, the solid-state imager 432 and the base substrate 431 maybe provided as a previously-manufactured unit and the remainder of thebiosensor 400 constructed thereon. In other embodiments, the providingoperation 502 includes manufacturing the base substrate 431 and thesolid-state imager 432 thereon using integrated circuit manufacturingprocesses. More specifically, the base substrate 431 and the solid-stateimager 432 may be manufactured by, for example, growing, depositing, andetching various layers and features in the layers (e.g., the lightdetectors 436). In particular, the solid-state imager 432 is an imagesensor that is manufactured using processes similar to complementarymetal-oxide-semiconductor (CMOS) technology. The remaining layers 433and 434 may also be manufactured using integrated circuit manufacturingprocesses.

At 504, a substrate material 520 may be provided onto the solid-stateimager 432 as shown in FIG. 9. The substrate material 520 may be grownor deposited onto the solid-state imager 432. In particular embodiments,the substrate material 520 includes glass or plastic. However,alternative materials that are capable of being formed as describedherein may also be used. With respect to FIG. 10, the method 500 mayalso include forming at 506 the filter walls 440 from the substratematerial 520. The forming operation 506 may include removing (e.g.,through etching) portions of the substrate material 520 to form thefilter walls 440. As shown in FIG. 10, chambers 522 may be formed at 506that are defined by the filter walls 440 and the adjacent layer 432,which is the solid-state imager 432 in the illustrated embodiment.

FIG. 10 shows a side cross-sectional view of the chambers 522. Thechambers 522 include a chamber opening 524 that provides access to thechamber 522 from the ambient environment or exterior 525 above. Thechambers 522 may constitute channels or recesses that extend from therespective chamber openings 524 to the solid-state imager 432. Across-section C of the chamber 522 taken perpendicular to the arrow inFIG. 10 may have various shapes. For example, the cross-section C may bepolygonal (e.g., rectangular, pentagonal, and the like) or substantiallycircular. In the illustrated embodiment, the cross-section of thechannel is square-shaped.

A coating 526 may be applied at 508 to the filter walls 440 as shown inFIG. 11. Various materials for the coating 526 may be used. Forinstance, when the biosensor 400 (FIG. 7) is fully constructed, thecoating 526 is configured to reflect light emissions that propagatethrough the chamber opening 524 toward the solid-state imager 432. Assuch, the coating 526 may be any material that is capable of beingcoated onto the filter wall 440 and that is also capable of reflectingthe light emissions. By way of example, the coating 526 may include ametal, such as aluminum. Alternatively, the coating 526 may be adielectric material having an index of refraction that is less than theindex of refraction of the light-absorbing material 446. The coating 526may extend entirely around the filter walls 440 as shown in FIG. 11 or,as will be described in greater detail below with respect to FIG. 16,the coating 526 may be located on only portions of the filter walls 440.Although not shown, during the applying operation 508 the coating 526may also be deposited onto the solid-state imager 432. In such cases,the coating 526 on the solid-state imager 432 may be removed (e.g.,through etching).

The method 500 also includes depositing or applying at 510 thelight-absorbing material 446 into the chambers 522 (FIG. 10). As shownin FIG. 12, the light-absorbing material 446 extends to a fill line 528that is substantially flush with ends 530 of the filter walls 440.However, as indicated by the arrows, the fill line 528 may have otherheights. For example, the fill line 528 may clear the ends 530 such thatthe ends 530 are located a depth within the light-absorbing material446. The fill line 528 may also be located a depth below the ends 530such that a portion of the chamber 522 remains unfilled. In suchembodiments, the unfilled portions of the chambers 522 may at leastpartially define reaction chambers as described herein.

The passivation layer 434 may be applied (e.g., deposited) at 512 ontothe light-absorbing material 446 as shown in FIG. 13. In suchembodiments in which an unfilled chamber portion remains after thedepositing operation 508, the applying operation 512 may includeinserting passivation material into the unfilled chamber portions sothat reaction chambers are formed. Such reaction chambers may be definedentirely by the passivation material or by a portion of the passivationmaterial and the filter walls 440 (e.g., the filter walls 440 may clearand project beyond the passivation layer 434). In other embodiments, theunfilled chamber portions may be filled entirely by the passivationmaterial so that the filter walls 440 are substantially flush with thepassivation layer 443.

In some embodiments, the method 500 may also include applying at 514 ashaped adhesive strip or layer 532 (shown in FIG. 14, also illustratedin FIG. 7 as the sidewalls as further described below) onto thepassivation layer 434. The flow cover 410 (shown in FIG. 15) may then bemounted to the adhesive layer 532. In FIG. 14, the adhesive layer 532 isa single sheet of material that is shaped to include an opening 534.However, in alternative embodiments, multiple layer sections may bedeposited onto the passivation layer 434 that effectively define theopening 534. With the flow cover 410 mounted onto the adhesive layer532, the adhesive layer 532 may then be cured or hardened to affix theflow cover 410 and the adhesive layer 532 to the passivation layer 434thereby forming the flow cell 402. The adhesive layer 532 defines thesidewalls 406, 408. In some embodiments, the adhesive layer 532comprises a photopolymer that is cured when exposed to ultraviolet orvisible light. As shown in FIG. 14, the adhesive layer 532 may have athickness T₁ that is substantially equal to the height H₁ of the flowchannel 418.

Optionally, the method 500 may also include providing at 518 thereaction sites 414 onto the passivation layer 434. Although theproviding operation 518 is shown in FIG. 8 as occurring after theoperations 514 and 516, the providing operation 518 may occur after orduring the application of the passivation layer 434. For example, theapplying operation 512 may include coupling the passivation layer 434onto the filter layer 433 (or another layer in alternative embodiments),wherein the passivation layer 434 includes previously fabricated padsthat are configured to have analytes-of-interest immobilized thereon. Inother embodiments, after the passivation layer 512 is applied (andbefore the adhesive layer and flow cover are coupled to the detectiondevice), the reaction sites or portions thereof may be patterned ontothe passivation layer 512. For example, the reaction sites may includepads or metal regions that are described in U.S. Provisional ApplicationNo. 61/495,266, filed on Jun. 9, 2011, and U.S. Provisional ApplicationNo. 61/552,712, filed on Oct. 28, 2011. Each of the U.S. ProvisionalApplication No. 61/495,266 (the '266 application) and the U.S.Provisional Application No. 61/552,712 (the '712 application) isincorporated herein by reference in its entirety. In some embodiments,the reaction sites 414 may be fabricated after the flow cell 402 ismanufactured on the detection device 404.

The incorporated '712 application describes techniques for preparing anarray of reaction sites. Exemplary techniques of the '712 applicationare illustrated by FIGS. 17-28. In some embodiments, each of thereaction sites is particularly suited for capturing a single molecule ofinterest at the reaction site. Once the molecule has been captured atthe reaction site, the molecule may be amplified to provide a pluralityof molecules (e.g., cluster) having the same chemical structure at thereaction site. For example, FIG. 17 generally represents certain phasesincluded in the preparation of the reaction sites on a detector surface,such as the detector surface 412. As described above, the passivationlayer 434 may be any one or more of various types of material that arecapable of shielding or protecting the circuitry from fluids andreactants used in an analytical detection procedure. For example, thepassivation layer 434 may include polymeric materials, plastics,silicon, quartz (fused silica), borosilicate glass (e.g., BOROFLOAT®borosilicate glass), sapphire, plastic materials.

At a site formation phase 830, reaction sites 832 (or individual sites)are formed on the passivation layer. The reaction sites 832 may belocated on the passivation layer so that each of the reaction sites 832is located with respect to an associated light detector (not shown inFIG. 17), such as the light detector 436. More specifically, a reactionsite 832 is located relative to the associated light detector such thata substantial portion of light emissions that propagate toward thedetector surface propagate through the detection path of the associatedlight detector and/or are incident upon the light detector. Asubstantial portion of light emissions may be greater than any otherportion of light emissions that are detected by other light detectors.In other words, a substantial portion is greater than crosstalk portionsdetected by non-associated light detectors.

A range of different techniques are presently contemplated for formationof the individual sites. One of these techniques is adapted to dispose amaterial at each site location that can be built upon for accommodatingthe molecule capture and amplification desired. Exemplary techniquesinclude nano-imprint lithography, described in greater detail below, aswell as dip pen lithography, photolithography, and micelle lithography.In one presently contemplated embodiment, the reaction sites are formedby deposition of a base pad at each site location. The site pads may bemade of any suitable material, such as gold or another metal. Othersuitable material may include silanes, functional biomolecules such asavidin or functionalized organic or inorganic molecules, titanium,nickel, and copper. Alternatively, the site pads can be created bysimply blocking the interstitial space with a resist or chemical moietythat resists attachment of a binding moiety leaving the site padcomposed of native substrate material (i.e. glass, etc). The site padscan then be derivatized with binding moieties that react specificallywith the substrate material (i.e. glass, etc.) and not interstitialspace. It should be noted that the array of base pads could be an arrayof nanodots or nanoparticles.

Once the sites are laid out on the passivation layer, site preparationmay proceed as indicated at reference numeral 834, resulting in aprepared microarray 836 ready to be further processed to receive asample of molecules to be tested. This phase of the manufacturingprocess may include deposition of various materials on the pads, butalso around the pads or over the entire extent of the passivation layer.These materials can be adapted to enhance the capture of a singlemolecule at each site location, and optionally for subsequentlyamplifying the molecules for further reading analysis.

It should also be noted that while biosensors have a single preparedfunctionalized surface as illustrated and described here, the biosensorsmay be used in applications where more than one functionalized surfaceis prepared and used for molecule captures, amplification, reading andanalysis. In some embodiments, the biosensors are configured to receivesolutions in the flow channel that permits the introduction of chemistryuseful for adding nucleotides and other substances, templates forreading, sequencing, and so forth, agents for deblocking locations onthe templates, washing and flushing liquids, and so forth. For example,U.S. patent application publication no. US 2010/0111768 A1 and U.S. Ser.No. 13/273,666, each of which is hereby incorporated by reference in itsentirety, describe similar protocols for flow cells. These protocols mayalso be used with the biosensors described herein.

Once the functionalized surface has been prepared and the flow cell hasbeen mounted to the detection device, the biosensor can be employed tocapture a single molecule at each site location as indicated by themolecule/capture phase 838 in FIG. 17. This single molecule willtypically be amplified, such as by bridge amplification, although otheramplification processes may also be used. For example, amplification ofa template nucleic acid can be carried out using bridge amplification asdescribed in U.S. Pat. Nos. 5,641,658 or 7,115,400; or in U.S. Pat. Pub.Nos. 2002/0055100 A1, 2004/0096853 A1, 2004/0002090 A1, 2007/0128624 A1,or 2008/0009420 A1, each of which is incorporated herein by reference inits entirety. In this example, the bridge amplification can be primed byprimer nucleic acids that are attached to a porous attachment layer thatis in contact with a base pad to which a template nucleic acid isattached. Thus, the base pad can seed growth of a cluster of nucleicacid copies of the template that forms in the porous attachment layeraround the base pad.

Another useful method for amplifying nucleic acids is rolling circleamplification (RCA). RCA can be carried out, for example, as describedin Lizardi et al., Nat. Genet. 19:225-232 (1998) or US Pat. Pub. No.2007/0099208 A1, each of which is incorporated herein by reference inits entirety. Also useful is multiple displacement amplification (MDA),for example, using a product of RCA (i.e. an RCA amplicon) as atemplate. Exemplary methods of MDA are described in U.S. Pat. Nos.6,124,120; 5,871,921; or EP 0,868,530 B1, each of which is incorporatedherein by reference in its entirety. In embodiments that include anamplification step, one or more primers that are used for amplificationmay be attached to a base pad or the porous attachment layer. Theprimers need not be attached to a base pad or a porous attachment layerin some embodiments.

A single molecule that is captured at a reaction site or otherwise usedin a method or composition herein can be a nucleic acid that is singlestranded or double stranded. Typically the nucleic acid will have asingle copy of a target sequence of interest. Nucleic acids havingconcatameric copies of a particular sequence can be used (e.g. productsof rolling circle amplification). However, in many embodiments thenucleic acid will not have concatameric copies of a sequence that is atleast 100 nucleotides long or that is otherwise considered a targetsequence for a particular application of the methods. Although themethods and compositions are exemplified with respect to capture of asingle nucleic acid molecule, it will be understood that other moleculesand materials such as those set forth above can also be captured at areaction site or otherwise used.

The prepared functionalized surface with the probes attached, asindicated by reference numeral 840, may then be used for analysispurposes. The reading/processing phase 842 is intended to include theimaging of the reaction sites on the functionalized surface, the use ofthe image data for analysis of the molecules captured and amplified ateach of the reaction sites, and so forth.

As mentioned above, one presently contemplated approach for forming thebase pads or site locations on substrate involves large-area patterningof very small features using techniques such as nanoscale imprintlithography. FIGS. 18-23 illustrate exemplary steps in an imprintlithography process. The lithography process may be performed using thepassivation layer (or other layers) as the passivation layer is on thedetection device 404. Alternatively, the lithography process may be usedto manufacture a layer (or layers) that is then subsequently mounted tothe detection device.

Referring first to FIG. 18, a substrate die 828 is first coated with atransfer layer 846, such as by spin or spray coating. In someembodiments, the substrate die 828 may be mounted onto the filter layer433, which may be mounted onto the substrate layers 432, 431. This layermay be formed of a commercially available resist, such as chlorobenzeneand a methylacrylate polymer, and may have a nominal thickness ofapproximately 70 nm. On this transfer layer, an ultraviolet (UV) imprintresist layer 848 is disposed. The resist layer also may be formed by apolymer which may be spin or spray coated on the transfer layer. This UVimprint resisted layer will form an etch barrier in subsequentprocessing. This layer may be formed, for example, of tert-butylmethylacrylate and polyester modified polydimethylsiloxane and polyesteracrylate and a photo-initiator, at a nominal thickness of approximately10 nm thicker than the feature height on a working mold 850, typically70 nm. The working mold 850 is formed in advance, and may be made ofvarious materials, such as glass or modified polydimethylsiloxane. Theworking mold will be generally transparent to UV light, to permit curingas described below. The desired pattern for the site pad will be formedin the working mold, such that recesses 852 will separate lands 854. Therecesses 852 will generally correspond to spaces that will be formedaround the pads on the substrate, while the lands 854 in this embodimentwill generally correspond to the locations of the pads. The size of theseparated lands can be tuned and can range, for example, from 5 nm(nanometers) to 3 μm (micrometers).

As illustrated in FIG. 19, during processing the mold is brought intocontact with the UV imprint layer and displaces portions of this layerto form regions 856 within the recesses 852 of the mold. That is, thelands 854 displace the UV imprint resist layer such that the lands aregenerally adjacent to the underlying transfer layer. With the mold inplace, then, the structure is exposed to UV radiation to at leastpartially cure the regions 856, rendering them resistant to subsequentetching and effectively transferring the pattern on the working moldinto the resist. With the mold then removed, as illustrated in FIG. 20,the transfer layer 846 remains on the substrate die 828, and theremaining regions 856 of the UV imprint resist layer remain to protectthe underlying regions of the transfer layer. Exposed transfer regions860 remain at what will become the locations of the site pads. An etchprocess is then used to remove these regions as illustrated in FIG. 21.Once the exposed transfer regions are removed, exposed substrate regions862 will remain. Subsequently, the structure is subjected to adeposition process, such as a metal deposition, to deposit a layer ofmaterial 864 over both the regions 856 and the exposed substrate regions862. In a currently contemplated embodiment, the deposition is of a thinlayer of gold, although other materials may include Al, Al₂O₃, Zn, ZnO,Ni, Ti, TiO₂, ITO (Indium tin oxide), etc. Moreover, the deposition maybe to any desired thickness, such as a nominal thickness of 5 nm.Finally, in a lift-off step, the layers above and below the regions 856,including these regions themselves are removed to leave only the pads atlocations 832 and the substrate die 828. This lift-off operation mayinvolve solvent washing steps and sonification. Following theseprocesses, a substrate die 828 will be provided with the sitesdetermined and formed in the desired pattern of sites, domains, regions,and so forth. The substrate die 828 may be the passivation layer 434described herein with respect to the detection device 404. The pads maybecome part of the reaction sites 414.

Once the sites are laid out and formed by positioning the site pads onthe substrate, subsequent building of the sites and preparation stepsmay take place. As illustrated in FIG. 24, in a presently contemplatedembodiment, each base pad 868 receives a capture substance 870 designedto promote the capture of a single molecule of interest. FIG. 24, aswith other figures in this disclosure, is not necessarily drawn toscale. For example, the capture substance can be submicroscopic in size(e.g. a linker molecule) or can be a particle that is, at least in somecases, visible under a microscope. In a presently contemplatedembodiment, the substance comprises thio-avidin, although othersubstances may be utilized, such as silanes, biotin-binding proteins,functional biomolecules such as avidin, streptavidin, neutravidin, andfunctionalized organic or inorganic molecules. An example is agold-patterned array functionalized with thiol-avidin to bind singlemolecules modified with biotin. Other capture substances may include,for example, biological binding molecules including neutravidin,streptavidin, antibodies, etc., chemical binding moieties such asamines, aldehydes, carboxyl groups, etc.; and inorganic binding moietiessuch as metal chelates (i.e. histidine binding), gold (thiol binding),etc.

A capture substance can be attached to a base pad or site via a covalentor non-covalent linkage. Exemplary covalent linkages include, forexample, those that result from the use of click chemistry techniques.Exemplary non-covalent linkages include, but are not limited to,non-specific interactions (e.g. hydrogen bonding, ionic bonding, van derWaals interactions etc.) or specific interactions (e.g. affinityinteractions, receptor-ligand interactions, antibody-epitopeinteractions, avidin-biotin interactions, streptavidin-biotininteractions, lectin-carbohydrate interactions, etc.). Exemplarylinkages are set forth in U.S. Pat. Nos. 6,737,236; 7,259,258; 7,375,234and 7,427,678; and US Pat. Pub. No. 2011/0059865 A1, each of which isincorporated herein by reference.

As illustrated in FIG. 25, then, in a presently contemplated embodimenta charged layer 872 may be disposed over the pads and capture substance.In this embodiment, if used, the charged layer comprisesaminopropyltriethoxysilane (APTES). This charged layer may promote theattachment of the single molecules at each site, while preventingattachment where not desired. As illustrated in FIG. 26, an attachmentlayer 74 is disposed over at least the pads 868, and in the illustratedembodiment may be disposed over the entire substrate. In otherembodiments, the attachment layer can be patterned such that it ispresent over the pads or sites but substantially absent overinterstitial regions between the pads or sites.

An attachment layer used in a method or composition herein may be formedof a micro-porous material, such as silane-free acrylamide (SFA).Silane-free acrylamide (SFA) polymer can be formed by polymerization ofsilane free acrylamide and N—(S bromoacetamidylpentyl) acrylamide(BRAPA). Other attachment layers that can be used include withoutlimitation, acrylamide, methacrylamide, hydroxyethyl methacrylate,N-vinyl pyrolidinone or derivatives thereof. Such materials are usefulfor preparing hydrogels. In some embodiments, the polymerizable materialcan include two or more different species of compound that form aco-polymer. Exemplary hydrogels and polymerizable materials that can beused to form hydrogels are described, for example, in US Pat. Pub. No.2011/0059865 A1, which is incorporated herein by reference in itsentirety. Other hydrogels include but are not limited to, polyacrylamidepolymers formed from acrylamide and an acrylic acid or an acrylic acidcontaining a vinyl group as described, for example, in WO 00/31148(incorporated herein by reference in its entirety); polyacrylamidepolymers formed from monomers that form [2+2] photo-cycloadditionreactions, for example, as described in WO 01/01143 or WO 03/014392(each of which is incorporated herein by reference in its entirety); orpolyacrylamide copolymers described in U.S. Pat. No. 6,465,178, WO01/62982 or WO 00/53812 (each of which is incorporated herein byreference in its entirety). The attachment layer can function to attachthe single molecules and/or it can provide locations for attachment ofidentical molecules (i.e. copies of the single molecules) at each siteduring amplification.

As noted above, various layouts may be envisaged for the sites of themicroarray. Moreover, the density, location, pitch, and sizes of thesites may vary depending upon such factors as the array design, the typeof processing and imaging equipment used for analyzing the arrays, andthe molecules to be processed. By way of example, presently contemplatedsites made as set forth in the present disclosure may have sizes ofapproximately 30-300 nm. The sites can be disposed on the substrate in ahexagonal pattern. The sites can be present at a density ofapproximately 1 million capture sites per square millimeter, but caneasily be tuned by adjusting the pitch to densities greater than 5million capture sites per square millimeter. While the particular pitchof the sites may vary, depending, for example, upon their size and thedensity desired, typical pitches may include at most about 5 micron, 2micron 1 micron, 850 nm or an even lower maximum value.

The sites or pads used in various embodiments can be in a size rangethat is useful for capture of a single nucleic acid template molecule toseed subsequent formation of a homogenous colony, for example, viabridge amplification. FIG. 27 illustrates a base pad 868 that isattached to a capture substance 870 that is in turn attached to a singlenucleic acid template 876. The nucleic acid template is illustrated asextending out of the attachment layer 874. However, in some embodimentsthe nucleic acid template can be retained under or within the volume ofthe attachment layer. Bridge amplification can be primed by primernucleic acids that are attached to the attachment layer (e.g. theattachment layer can be a gel) to seed growth of a cluster of nucleicacid copies of the template that forms in or on the attachment layeraround the base pad 868.

In an exemplary bridge amplification method, a template nucleic acidhybridizes to a gel-attached primer and the 3′ end of the primer isextended to create a complementary copy of the template. In someembodiments two different primers can be attached to the gel. Theprimers can form a pair used for amplification of a template and itscomplementary copy. As such, two primers can be used for amplificationof the template into multiple copies to form a nucleic acid cluster orpopulation of amplicons. For example, amplification can be carried outusing bridge amplification to form nucleic acid clusters attached to thegel. Useful bridge amplification methods are described, for example, inU.S. Pat. Nos. 5,641,658 and 7,115,400; U.S. Pat. Pub. Nos. 2002/0055100A1, 2004/0096853 A1, 2004/0002090 A1, 2007/0128624 A1, and 2008/0009420A1, each of which is incorporated herein by reference in its entirety.Other useful methods for amplifying nucleic acids using one or moregel-attached primers are rolling circle amplification (RCA) and multipledisplacement amplification (MDA).

In particular embodiments, a cluster of nucleic acids may have a footprint that is no larger than the area of the base pad. For example, theattachment layer 874 may be confined to the foot print of the base pad868. As such the base pad (and optionally the attachment layer) can forma cluster restriction zone along the lines illustrated in FIG. 28.Alternatively, the foot print of a cluster may be larger than the basepad 868 from which it was seeded.

The incorporated '266 application also describes various methods ofpreparing a detector surface having a designated pattern of reactionsites. At least some of these methods may be used to pattern thedetector surfaces of the exemplary biosensors described herein. Forexample, the surface of the passivation layer may have an array ofdiscrete metal regions in which each metal region is surrounded byinterstitial region(s), such as glass. The metal regions may befabricated on the surface of the passivation layer by etching orphotolithographic processes. The metal regions may be, for example,aluminum oxide or gold. The metal regions may be used to form thereaction sites having analytes-of-interest as described herein. Inparticular embodiments, the metal regions have clusters of nucleic acidsthereon.

FIG. 29 is an enlarged cross-section of a portion of a biosensor 538,which may have similar features as the biosensor 400 (FIG. 7). Forexample, the biosensor 538 includes a passivation layer 539, a filterlayer 540, and a solid-state imager 542 having light detectors 536.Similar to the filter layer 433 (FIG. 7), the filter layer 540 mayinclude filter walls 550 and a light-absorbing material 552 extendingbetween adjacent filter walls 550. In the illustrated embodiment, thefilter walls 550 have a height H₂ and the light-absorbing material 552has a thickness T₂ that is substantially equal to the H₂. However, inalternative embodiments, the thickness T₂ may be less than the height H₂such that reaction chambers may be defined, or the thickness T₂ may begreater than the height H₂ such that ends 554 of the filter walls 550are located a depth into the light-absorbing material 552.

The filter walls 550 may include first and second interior portions 562and 564 in which the first interior portion 562 is coated with a coatingmaterial 566 and the second interior portion 564 is not coated or iscoated with a different material. In the illustrated embodiment, thecoating material 566 is a reflective material. In an exemplaryembodiment, the first interior portion 562 is coated with a materialthat includes aluminum or another metal and has a thickness T₃ that is,at most, about 3000 A. In some embodiments, the thickness T₃ of thecoating material 566 is less than about 2000 A or less than about 1000A. In more particular embodiments, the coating material 566 has athickness T₃ that is less than about 600 A. In some embodiments, thethickness T₃ of the coating material 566 tapers or decreases as thecoating material 566 extends closer to the solid-state imager 542. Forexample, the thickness T₃ may be about 600 A near an end 554 of thefilter wall 550 that is proximate to the passivation layer 539. However,the thickness T₃ may decrease to zero as the coating material 566extends toward the solid-state imager 542.

As shown, a detection path 560A extends between the reaction site 556Aand the light detector 536A, and another detection path 560B extendsbetween the reaction site 556B and the light detector 536B. A detectionpath represents the general space or volume of the biosensor 538 thatlight (e.g., emission light, excitation light, and crosstalk light)propagates through from one reaction site 556 to an associated lightdetector 536. The detection paths 560A, 560B extend through thepassivation layer 539, the filter layer 540, and a portion of thematerial in the solid-state imager 542.

In FIG. 29, the reaction sites 556A, 556B may be characterized as beingadjacent to each other. Likewise, the light detectors 536A, 536B may becharacterized as being adjacent to each other. Also, the light detector536A is positioned with respect to the filter walls 550 and the reactionsite 556A to detect emission light from the reaction site 556A. As such,the light detector 536A and the reaction site 556A are characterized asbeing associated with each other. Similarly, the light detector 536B andthe reaction site 556B are characterized as being associated with eachother because the light detector 536B is configured to detect emissionlight from the reaction site 556B.

Also shown in FIG. 29, the detection paths 560 may have a changing orreducing width. The width for each of the detection paths 560 may beginto decrease at some point as the detection path 560 extends toward thesolid-state imager 542. For example, the filter walls 550 may have anincreasing thickness as the filter walls 550 extend from the ends 554toward the solid-state imager 542. For example, the ends 554 may have athickness TL that is between about 0.4 microns to about 1.2 microns. Inparticular embodiments, the thickness TL is about 0.8 microns at theends 554. As shown in FIG. 13, the thickness TL may begin to increase asthe filter wall 550 transitions from the first interior portion 562 tothe second interior portion 564. The filter walls 550 may have athickness TG at the solid-state imager 542. Consequently, as thethickness increases, the width decreases from W_(G) to about W_(L). Inthe illustrated embodiment, the light detectors 536 have a width W_(D)that is about equal to 1.4 microns. However, the light detectors 536 maybe less than or more than 1.4 microns in other embodiments.

Example light rays 570 that are emitted from the reaction sites 556 areshown in FIG. 29. Light emissions may propagate from the reaction site556 or, more specifically, from the analyte-of-interest from thereaction site 556 in an isotropic manner. Accordingly, at least some ofthe light rays 570 are transmitted through the detection paths 560A,560B toward the light detectors 536. As shown in FIG. 29, the light rays570 that are reflected by the coating 566 may be less attenuated thanthe light rays 570 that are reflected by the second interior portion 564of the filtered walls 550. In such embodiments, quantum efficiency (QE)of the light detected by the light detectors 536 may be improved.

FIG. 30 illustrates a detection device 600 having a plurality ofreaction sites 656A-656D on a detector surface 602. The detection device600 includes light detectors 636A-636D and may be similar to otherdetection devices described herein. The light detectors 636A-636D areassociated with the reaction sites 656A-656D, respectively.Corresponding detection paths 640A-640D extend between the lightdetectors 636A-636D and corresponding reaction sites 656A-656D. Thearrows that indicate the detection paths 640A-640D are merely toillustrate a general direction that the light propagates through therespective detection path.

During an imaging event, the detection device 600 is configured todetect light using the light detectors 636A-636D. As demonstrated inFIG. 30 by pyramidal hash marked areas or zones, light emissions (oremission signals) are propagating from the reaction sites 656A and 656B,but light emissions are not propagating from 656C or 656D. The lightemissions may be indicative of, for example, a positive binding eventbetween the analytes-of-interest located at the corresponding reactionsite and another biomolecule. In particular embodiments, the reactionsites 656A-656D are illuminated by an excitation light (e.g., 532 nm).The reaction sites 656A and 656B are bound to respective biomoleculeshaving light labels (e.g., fluorescent moieties). In response to theexcitation stimulus, the reaction sites 656A and 656B provide lightemissions as demonstrated in FIG. 30.

However, the reaction sites 656 and the light detectors 636 may belocated relatively close to one another such that light emissions from anon-associated reaction site may be detected by a light detector. Suchlight emissions may be referred to as crosstalk emissions. By way ofexample, the light emissions propagating from the reaction site 656Ainclude a crosstalk portion and a site portion. The site portion of thelight emissions from the reaction site 656A is that portion of the lightemissions that is configured to be detected by the light detector 636A.In other words, the site portion includes the light emissions thatpropagate at an angle that is generally toward the light detector 636Asuch that filter walls 630 defining the detection path 640A are capableof directing the light emissions toward the light detector 636A. Thecrosstalk portion is that portion of the light emissions that clears thefilter walls 630 defining the detection path 640A and propagates into,for example, the detection path 640B. In such cases, the crosstalkportion may be directed to the light detector 636B, which is notassociated with the reaction site 656A. Thus, the light detector 636Bmay be referred to as a non-associated light detector with respect tothe reaction site 656A.

Using the embodiment shown in FIG. 30 as an example, the light detector636A may detect the site emissions from the reaction site 656A and thecrosstalk emissions from the reaction site 656B. Likewise, the lightdetector 636B may detect the site emissions from the reaction site 656Band the crosstalk emissions from the reaction site 656A. The lightdetector 636C may detect the crosstalk emissions from the reaction site656B. However, the reaction site 656C is not providing light emissionsin FIG. 30. Thus, an amount of light detected by the light detector 636Cis less than the corresponding amounts of light detected by the lightdetectors 636A and 636B. As shown in FIG. 30, the light detector 636Conly detects crosstalk emissions from the reaction site 656B, and thelight detector 636D does not detect crosstalk emissions or siteemissions.

FIG. 31 is a top plan view of the detector surface 602 during first,second, and third imaging events. The detector surface 602 includes aset of the reaction sites 656. The reaction sites 656 are labeled byrow-letter and column-number. In the illustrated embodiment, thereaction sites 656 remain in the same position or location for themultiple imaging events. The detector surface 602 may include aplurality of image areas 610 that are defined by the filter walls 630.In FIG. 31, the image areas 610 are substantially square-shaped, butother shapes may be implemented. In the illustrated embodiment, each ofthe image areas 610 includes only a single reaction site 656 that coversa site area 612. The site area 612 may be substantially circular asshown in FIG. 31 or may have other shapes. Furthermore, the site areas612 are not required to have the same or similar shapes. In FIG. 31, thesite area 612 is substantially less than the corresponding image areas610. However, in other embodiments, the site area 612 may be slightlyless than, substantially equal to, or slightly greater than thecorresponding image area 610. In such embodiments in which the site area612 is greater than the image area 610, adjacent image areas 610 may beseparated by greater distances to accommodate the site areas 612. Forexample, the filter walls 630 may be configured to be thicker.Alternatively, the detector surface 602 may be configured so thatinactive image areas 610 that do not have a corresponding reaction site656 separate active image areas 610 that do have a reaction site 656.

In some embodiments, the reaction sites 656 may not have a commonlocation with respect to the filter walls 630 that define the detectionpath. This may be due to, for example, tolerances in the manufacturingor sample preparation processes. For example, as shown in FIG. 31, thereaction site 656 at E1 is substantially centered between four filterwalls 630, but the reaction site 656 at E2 is closer to walls 630A and630B than walls 630C and 630D. During different imaging events, adifferent number of reaction sites may emit light that is indicative ofa desired reaction (e.g., binding event). In FIG. 31, the darker-shadedreaction sites 656 (or site areas 612) indicate that the reaction site656 is providing emission signals during the corresponding imagingevent, and the non-shaded and hashed reaction sites 656 (or site areas612) indicate that the reaction site 656 is not providing emissionsignals during the imaging event. For instance, the reaction sites E2,F2, and G3 are emitting light during the first imaging event; thereaction sites E2, E3, F1, G1, G2, and G3 are emitting light during thesecond imaging event; and the reaction sites E1, F1, F2, F3, G1, and G3are emitting light during the third imaging event.

Embodiments described herein may be configured to account for crosstalkemissions from adjacent reaction sites when analyzing signal data fromthe detection device. For example, each light detector 636 (FIG. 30) maybe assigned a crosstalk function that is based on whether adjacentreaction sites are providing light emissions. In some cases, thecrosstalk function may be based on an amount of light emitted from theadjacent site(s), which may be determined by an amount of light detectedby the light detector(s) that is/are associated with the adjacentsite(s). For example, the reaction site 656 at F1 during the thirdimaging event is providing emission signals and the reaction sites atE1, F2, and G1 are also providing emission signals. Thus, a substantialportion of an amount of light detected by the light detector associatedwith the reaction site 656 at F1 may be due to crosstalk emissions fromthe reaction sites at E1, F2, and G1.

As another example, the reaction site 656 at F2 during the secondimaging event does not provide light emissions. However, six adjacentreaction sites 656 (E2, E3, F1, G1, G2, and G3) may provide crosstalkemissions. Thus, during the second imaging event, an entire portion oflight detected by the light detector associated with the reaction site656 at F2 is due to crosstalk emissions from the reaction sites 656 atE3, E3, F1, G1, G2, and G3. The cumulative effect of the crosstalkemissions from the reaction sites 656 at E2, E3, F1, G1, G2, and G3 mayresult in an incorrect determination as to whether a binding eventoccurred at the reaction site of F2.

FIG. 32 illustrates exemplary light scores or values obtained by thelight detectors that are associated with the reaction sites 656 shown inFIG. 31. In the illustrated embodiment, the light scores range from5-105. However, these are only illustrative and other ranges may be usedor other manners for indicating a light score. As shown, light scoresobtained by light detectors may be substantially based uponnon-associated reaction sites as well as the associated reaction sites.For example, the light score for F2 during the second imaging event is60 even though the reaction site 656 at F2 did not provide emissionsignals. During the third imaging event, both of the reaction sites 656at F2 and G3 provide emission signals, but the light score associatedwith the reaction site 656 at F2 is greater than the light scoreassociated with the reaction site 656 at G3 due to crosstalk emissions.

FIG. 33 shows a method of 700 analyzing signal data obtained from adetection device, such as the detection devices described above. Themethod may be used to analyze light emissions (or emission signals) fromreaction sites that have a fixed position with respect to the associatedlight detectors of the detection device and that are exposed to astimulus (e.g., excitation light) multiple times (i.e., for separateimaging events). The method 700 may be performed by, for example, theanalysis module 138 (FIG. 2). The method 700 may include obtaining at702 signal data from the light detectors. The signal data may includelight scores, such as those shown in FIG. 32, that are based on anamount of light detected by the light detectors during an imaging event.The amount of light detected by the light detectors may include siteemissions and crosstalk emissions. More specifically, the amount oflight detected by one light detector may be based on whether crosstalkemissions from non-associated reaction site(s) are also detected by thecorresponding light detector. The amount of light detected by the lightdetector may also depend upon other factors, such as excitation light,manufacturing tolerances, electrical noise in the detection device, etc.

The method 700 also includes analyzing at 704 the light scores from aset or group of light detectors from a plurality of the imaging eventsto determine at 706 respective crosstalk functions of the lightdetectors. The group of light detectors may be an array or sub-array ofa detection device. The group of light detectors may be proximate to oneanother (e.g., one light detector may be adjacent to or within theimmediate vicinity of other light detectors). For instance, the group oflight detectors may be the light detectors associated with the reactionsites 656 shown in FIG. 31.

In some embodiments, the crosstalk function may be determined byanalyzing a relationship between a light score that is associated withone reaction site (i.e., site-of-interest) and the light scores that areassociated with adjacent reaction sites. For example, it may be assumedthat a light score of the reaction site 656 at G2 is dependent uponwhether the reaction site 656 at G2 provided light emissions (i.e., siteemissions) and whether the adjacent reaction sites 656 at F1, F2, F3,G1, and G3 also provided light emissions (i.e., crosstalk emissions).More specifically, the light score may be based on (a) the siteemissions from the reaction site 656 at G2 (if any) and (b) thecrosstalk emissions from the adjacent reaction sites 656 at F1, F2, F3,G1, and G3. By way of example, during the first imaging event, thereaction site 656 at G2 has a light score of 15. The reaction site 656at G2 did not provide light emissions, which may indicate that a bindingevent did not occur at the G2 prior to or during the imaging event.Thus, the value of the light score is based on the crosstalk portionsfrom the adjacent reaction sites 656 at F1, F2, F3, G1, and G3 thatactually provided light emissions. Whether or not a reaction siteprovides light emissions can be determined by identifying whether thereaction site in question had a light score that exceeded a designatedvalue (e.g., 70). In this example, the reaction sites 656 at F2 and G3provide light emissions during the first imaging event. Other methodsmay be used to determine whether a reaction site provides lightemissions.

However, the reaction site 656 at G2 during the third imaging eventreceived a light score of 40. Like the first imaging event, both of thereaction sites 656 at F2 and G3 provide light emissions. However, unlikethe first imaging event, the reaction sites 656 at F1, F3, and G1 alsoprovide light emissions. Thus, it may be assumed that the differencebetween the light scores of the first and third imaging events (i.e., 15and 40) for the reaction site 656 at G2 may be based on the crosstalkportions from the reaction sites 656 at F1, F3, and G1. By analyzing thelight scores of the light detectors in a similar manner for multipleimaging events, a crosstalk function for each of the light detectors maybe determined. In some embodiments, the crosstalk function is determinedby a total number of adjacent reaction sites that provide emissionssignals. For example, for each adjacent reaction site that providesemission signals (e.g., had a light score of 70 or greater), apredetermined value may be added to the crosstalk function for thesite-of-interest. In some embodiments, the crosstalk function may bebased not only on the number of adjacent reaction sites, but also theparticular combination of adjacent reaction sites. In this otherembodiment, the crosstalk function may be affected by the location ofthe adjacent reaction site(s).

After determining the crosstalk functions, the method 700 may alsoinclude analyzing at 708 the signal data for each of the imaging eventsusing the assigned crosstalk functions to determine characteristics ofthe analytes-of-interest. For instance, the crosstalk function of onelight detector may be subtracted from the light score of that lightdetector to determine a light score that is based primarily on a siteemissions (if any). As one example, if a light detector has a lightscore of 100, but the crosstalk function finds that 30 of the 100 aredue to adjacent reaction sites, then the modified (or more accurate)light score is 70. The modified light score may then be used todetermine a characteristic of the analyte-of-interest located at thecorresponding reaction site. In one specific application, thecharacteristic of the analyte-of-interest that is determined is thenucleotide that was recently incorporated into a sequence at thereaction site.

FIG. 34 is a top plan view of a detector surface 720 having a pluralityof reaction sites 722. As shown, in some embodiments, the reaction sites722 may be positioned with respect to each other to reduce any effectthat crosstalk emissions may have on light detection. For instance, atleast portions or areas of the detector surface 722 may be unused sothat distances between adjacent reaction sites become greater. These maybe the inactive image areas described herein. As one example, thepattern or configuration of the reaction sites 722 along the detectorsurface 720 in FIG. 34 is a “checkerboard” type configuration. In suchembodiments, each reaction site is adjacent to, at most, four otherreaction sites. However, the checkerboard configuration is only oneexample and other configuration may be used to control or reduce theeffects of crosstalk.

FIG. 35 illustrates a plan view of a variety of exemplary flow cellconfigurations 751-758 that may be used with one or more embodiments. Aninlet port 759 and an outlet port 760 are indicated as circles in theflow cells 751-758. Flow of solution is indicated by arrows labeled F.Embodiments described herein may use flow cells that are sized andshaped to be suitable for an intended purpose. For example, the flowcells may be configured to control flow of the solutions that are movedalong the detector surface of the detection device. Controlling flow ofa solution may also include controlling bubble formation and bubbledisposal. As shown, the flow cells 751-753 may have a single largeactive area such that the flow of solution does not turn, curve, or bendbetween the ports 759, 760. However, the flow cells 754-758 may have atleast one bend where a direction of flow is curved.

Embodiments described herein may also be used with various detectionprotocols. For example, U.S. Provisional Application No. 61/538,294,filed Sep. 23, 2011, which is incorporated by reference in its entirety,describes methods and systems that utilize fewer detection moieties thanthe number of analytes targeted for detection. For example, fordetecting the incorporation of four analytes (e.g., during a sequencingreaction) each of the analytes can be differentially labeled anddetected by one of four excitation/emission filters (e.g., fluorescentsequencing). Alternatively, methods and systems can also be utilizedwherein one dye, or a plurality of dyes with similar detectioncharacteristics, are used when detecting and differentiating multipledifferent analytes. As such, the number of detection moieties utilizedis less than the number of analytes being detected which can also serveto reduce the number of imaging events needed to determine the presenceof the different analytes. U.S. application Ser. No. 13/624,200, whichwas filed on Sep. 21, 2012, is also incorporated by reference in itsentirety.

FIG. 36 illustrates a cross-section of a biosensor 800 formed inaccordance with one embodiment. The biosensor 800 may have similarfeatures as the biosensor 102 (FIG. 1) and the biosensor 400 (FIG. 7)described above and may be used in, for example, the cartridge 302 (FIG.4). As shown, the biosensor 800 may include a flow cell 802 that ismounted onto a detection device 804. In the illustrated embodiment, theflow cell 802 is affixed directly to the detection device 804. However,as described in other embodiments, the flow cell 802 may be removablycoupled to the detection device 804. The detection device 804 has adetector surface 812 that may be functionalized (e.g., chemically orphysically modified in a suitable manner for conducting the desiredreactions). For example, the detector surface 812 may be functionalizedand may include a plurality of reaction sites 814 having one or morebiomolecules immobilized thereto. In particular embodiments, thereaction sites 814 include clusters or colonies of biomolecules (e.g.,oligonucleotides) that are immobilized on the detector surface 812. Suchreaction sites may be particularly suitable for SBS sequencing.

In particular embodiments, the detector surface 812 is prepared ormodified as described in U.S. application Ser. No. 13/784,368, filedMar. 4, 2013, and entitled “Polymer Coatings,” which is incorporated byreference in its entirety.

In the illustrated embodiment, the flow cell 802 includes sidewalls 806,808 and a flow cover 810 that is supported by the sidewalls 806, 808.The sidewalls 806, 808 are coupled to the detector surface 812 andextend between the flow cell cover 810 and the sidewalls 806, 808. Insome embodiments, the sidewalls 806, 808 are formed from a curableadhesive layer that bonds the flow cover 810 to the detection device804.

The sidewalls 806, 808 are sized and shaped so that a flow channel 818exists between the flow cell cover 810 and the detection device 804. Insome cases, the dimensions and the shape of the flow channel 818 may beconfigured to control bubble formation. As shown, the flow channel 818may include a height H₃ that is determined by the sidewalls 806, 808.The height H₃ may be between about 50-400 μm (microns) or, moreparticularly, about 80-200 μm. In particular embodiments, the height H₃may be between about 80-120 μm. In the illustrated embodiment, theheight H₃ is about 100 μm. The flow cover 810 may include a materialthat is transparent to excitation light 801 propagating from an exteriorof the biosensor 800 into the flow channel 818. As shown in FIG. 36, theexcitation light 801 approaches the flow cell cover 810 at anon-orthogonal angle. However, this is only for illustrative purposes asthe excitation light 801 may approach the flow cover 810 from differentangles. In some cases, the excitation light 801 floods the flow channel818.

Also shown, the flow cell cover 810 may include inlet and outlet ports820, 822 that are configured to fluidically engage other ports (notshown). For example, the other ports may be from the cartridge 302 (FIG.4) or the workstation 300 (FIG. 4). The flow channel 818 is sized andshaped to direct a fluid along the detector surface 812. The height H₃and other dimensions of the flow channel 818 may be configured tomaintain a substantially even flow of a fluid along the detector surface812. The dimensions of the flow channel 818 may also be configured tocontrol bubble formation.

As shown in exemplary FIG. 36, the sidewalls 806, 808 and the flow cover810 are separate components that are coupled to each other. Inalternative embodiments, the sidewalls 806, 808 and the flow cell cover810 may be integrally formed such that the sidewalls 806, 808 and theflow cell cover 810 are formed from a continuous piece of material. Byway of example, the flow cell cover 810 (or the flow cell 802) maycomprise a transparent material, such as glass or plastic. The flow cellcover 810 may constitute a substantially rectangular block having aplanar exterior surface and a planar inner surface that defines the flowchannel 818. The block may be mounted onto the sidewalls 806, 808.Alternatively, the flow cell 802 may be etched to define the flow cellcover 810 and the sidewalls 806, 808. For example, a recess may beetched into the transparent material. When the etched material ismounted to the detection device 804, the recess may become the flowchannel 818.

The detector surface 812 may be substantially planar as shown in FIG.36. However, in alternative embodiments, the detector surface 812 may beshaped to define reaction chambers in which each reaction chamber hasone or more of the reaction sites 814. The reaction chambers may bedefined, for example, by chamber walls that effectively separate thereaction site(s) 814 of one reaction chamber from the reaction site(s)814 of an adjacent reaction chamber.

The detection device 804 may be similar to, for example, an integratedcircuit comprising a plurality of stacked substrate layers 831-834. Thesubstrate layers 831-834 may include a base substrate 831, a solid-stateimager 832 (e.g., CMOS image sensor), a filter or light-management layer833, and a passivation layer 834. It should be noted that the above isonly illustrative and that other embodiments may include fewer oradditional layers. Moreover, each of the substrate layers 831-834 mayinclude a plurality of sub-layers. For example, the substrate layer 834includes layers (or sub-layers) 871-874. The detection device 804 may bemanufactured using processes that are similar to those used inmanufacturing integrated circuits, such as CMOS image sensors and CCDs.For example, the substrate layers 831-834 or portions thereof may begrown, deposited, etched, and the like to form the detection device 804.

The passivation layer 834 is configured to shield the filter layer 833from the fluidic environment of the flow channel 818. In some cases, thepassivation layer 834 is also configured to provide a solid surface(i.e., the detector surface 812) that permits biomolecules or otheranalytes-of-interest to be immobilized thereon. For example, each of thereaction sites 814 may include a cluster of biomolecules that areimmobilized to the detector surface 812. Thus, the passivation layer 834may be formed from a material that permits the reaction sites 814 to beimmobilized thereto. The passivation layer 834 may also comprise amaterial that is at least transparent to a desired fluorescent light. Byway of example, the passivation layer 834 may include silicon nitride(Si₃N₄) and/or silica (Sift). However, other suitable material(s) may beused. In the illustrated embodiment, the passivation layer 834 may besubstantially planar. However, in alternative embodiments, thepassivation layer 834 may include recesses, such as pits, wells,grooves, and the like. In the illustrated embodiment, the passivationlayer 834 has a thickness that is about 150-300 nm and, moreparticularly, about 175-250 nm.

In particular embodiments, the passivation layer 834 includes thesub-layers 871-874. The sub-layer 871 may be a stress-matching layerthat includes silica (Sift). The stress-matching layer 871 is configuredto support the materials deposited thereon and reduce the likelihood ofcracking of the passivation layer 834 and/or other layers. The sub-layer872 may be a protection layer that includes silicon nitride (Si₃N₄). Thesub-layer 873 may be a chemical-matching layer including silica (SiO₂).The chemical-matching layer 873 may be configured to receive asample-receiving layer 874. The sample-receiving layer 874 may beconfigured to have samples immobilized thereon. By way of example only,the sub-layers 871-873 may have thicknesses of about 25 nm, about 150nm, and about 25 nm, respectively. Stress of each sub-layer can beadjusted during deposition so that the overall layer has a desiredbalance to minimize winkling and bulking effects.

The filter layer 833 may include various features that affect thetransmission of light. In some embodiments, the filter layer 833 canperform multiple functions. For instance, the filter layer 833 may beconfigured to (a) filter unwanted light signals, such as light signalsfrom an excitation light source; (b) direct emission signals from thereaction sites 814 toward corresponding light detectors 836 that areconfigured to detect the emission signals from the reaction sites; or(c) block or prevent detection of unwanted emission signals fromadjacent reaction sites. As such, the filter layer 833 may also bereferred to as a light-management layer. In the illustrated embodiment,the filter layer 833 has a thickness that is about 1-10 μm and, moreparticularly, about 3-6 In the illustrated embodiment, the filter layer833 is about 4.0 μm.

In some embodiments, the filter layer 833 may include a plurality offilter walls 840. The filter walls 840 may be configured to at least oneof (a) reflect emission signals or (b) block or prevent unwantedemissions signals from adjacent reaction sites. Adjacent filter walls840 may define the detection path 845 for the emission signals that areprovided by one or more of the reaction sites 814. The detection path845 extends between the detector surface 812 to a light detector 836.The filter walls 840 may be formed from various kinds of materials. Forexample, the filter walls 840 may include an internal material 842(e.g., glass) and an exterior coating 844 that is deposited ontosurfaces of the internal material 842. In the illustrated embodiment,the coating 844 includes a reflective metal (e.g., aluminum). Inalternative embodiments, the coating 844 may include a dielectricmaterial.

The filter walls 840 may be similar to the filter walls 550 (FIG. 29).For example, the filter walls 840 have a height H₄ and a light-absorbingmaterial 846 has a thickness T₄ that is greater than the height H₄ ofthe filter walls 840. In an exemplary embodiment, the coating material844 includes aluminum or another metal and has a thickness T₅ that is,at most, about 3000 Angstroms (Å) (or 300 nm). In some embodiments, thethickness T₅ of the coating material 844 is less than about 2000 A (or200 nm) or less than about 1000 A (100 nm). In more particularembodiments, the thickness T₅ is less than about 600 A (60 nm). In someembodiments, the thickness T₅ of the coating material 844 tapers ordecreases as the coating material 844 extends closer to the solid-stateimager 832. For example, the thickness T₅ may be about 600 A near an end855 of the filter wall 840 that is proximate to the passivation layer834. However, the thickness T₅ may decrease to zero as the coatingmaterial 844 extends toward the solid-state imager 832.

Also shown in FIG. 36, the detection paths 845 between adjacent filterwalls 840 may have a changing or reducing width W. The width W for eachof the detection paths 845 may begin to decrease at some point as thedetection path 845 extends toward the solid-state imager 832 between theadjacent filter walls 840. For example, the filter walls 840 may have anincreasing thickness as the filter walls 840 extend from the ends 855toward the solid-state imager 832. For example, the ends 855 may have athickness TL that is between about 0.2 microns to about 1.2 microns. Inparticular embodiments, the thickness TL may be between about 0.2microns and about 0.4 microns. The filter walls 840 may have a thicknessTG at the solid-state imager 832. Consequently, as the thicknessincreases, the width W decreases. In the illustrated embodiment, thelight detectors 836 have a center-to-center spacing S_(LD) that is aboutequal to 1.4 microns. However, the center-to-center spacing S_(LD)between the light detectors 836 may be less than or more than 1.4microns in other embodiments.

The light-absorbing material 846 may be deposited over, matched to, orunder the filter walls 840. The light-absorbing material 846 mayinclude, for example, a material that is configured to absorb theexcitation light and permit the fluorescent emissions (i.e., emissionlight, emission signals) to pass therethrough. In the illustratedembodiment, the light-absorbing material 846 may comprise a resist-basedabsorption material that is configured to block, for example, 532 nmexcitation light. However, other light-absorbing materials 846 may beused. In alternative embodiments, a dichroic filter may be positionedbetween adjacent filter walls 840. In other alternative embodiments, adichroic filter layer is located above or below the filter walls 840.For example, the dichroic filter layer may be located between thepassivation layer 834 and the filter layer 833.

In alternative embodiments, the filter layer 833 may include an array ofmicrolenses or other optical components. Each of the microlenses may beconfigured to direct emission signals from an associated reaction site814 to an associated light detector 836. Such microlenses may be used inaddition to or as an alternative to the filter walls 840.

In some embodiments, the solid-state imager 832 and the base substrate831 may be provided together as a previously constructed solid-stateimaging device (e.g., CMOS chip). For example, the base substrate 831may be a wafer of silicon and the solid-state imager 832 may be mountedthereon. The solid-state imager 832 includes a layer of semiconductormaterial (e.g., silicon) and the light detectors 836. In someembodiments, each light detector 836 is formed from a single pixel. Inother embodiments, multiple pixels (e.g., 2, 3, 4, 5, 6, or more) mayform a single light detector 836. In the illustrated embodiment, thelight detector 836 pixels are photodiodes configured to detect light.

The solid-state imager 832 may include a dense array of light detectors836 that are configured to detect activity indicative of a desiredreaction from within or along the flow channel 818. In some embodiments,each light detector 836 has a detection area that is less than about 50μm². In particular embodiments, the detection area is less than about 10μm². In more particular embodiments, the detection area is about 2 μm².In such cases, the light detector 836 may constitute a single pixel. Anaverage read noise of each pixel in a light detector 836 may be, forexample, less than about 150 electrons. In some embodiments, the readnoise may be less than about 50 electrons or less that about 25. Inparticular embodiments, the read noise may be less than about 5electrons. The resolution of the array of light detectors 836 may begreater than about 0.5 megapixels (Mpixels). In more specificembodiments, the resolution may be greater than about 5 Mpixels and,more particularly, greater than about 10 Mpixels.

In some embodiments, the detection device 804 includes a microcircuitarrangement, such as the microcircuit arrangement described in U.S. Pat.No. 7,595,883, which is incorporated herein by reference in theentirety. More specifically, the detection device 804 may comprise anintegrated circuit having a planar array of the light detectors 836.Circuitry formed within the detection device 804 may be configured forat least one of signal amplification, digitization, storage, andprocessing. The circuitry may collect and analyze the detectedfluorescent light and generate data signals for communicating detectiondata to a bioassay system. The circuitry may also perform additionalanalog and/or digital signal processing in the detection device 804. Thecircuitry may include conductive pathways (e.g., wires) 850 thattransmit the data signals to substrate layer 831. The conductivepathways 850 may continue from the substrate layer 831 to, for example,a computing system. The conductive pathways may include, for example,electrical contacts 854 or other form of a PCB, input/output (I/O)interface.

Experimental results were performed using a biosensor having featuresdescribed herein, such as the embodiment shown and described withrespect to FIG. 36. An assay was performed using a PhiX DNA control forSBS sequencing using reversible terminators. U.S. application Ser. No.13/624,200, which is incorporated by reference in its entirety,describes methods and materials (e.g., dyes) that may be used with thebiosensor. The protocol used to obtain the experimental results issimilar to the PhiX protocol described in the “MiSeq System User Guide”from Illumina, Inc. (Part #15027617 Rev. G), January 2013, which isincorporated by reference in its entirety. The protocol was modified toinclude proprietary reagents that are provided in Miseq Reagent Kit v2.The modified protocol is described in greater detail in the “Preparing aPhiX Control for a Run on the MiSeq,” (Part #15036175 Rev. A) (2013),which is incorporated by reference in its entirety.

In one operating session, about 875,123 clusters were produced within aflow channel and about 794,873 of these clusters were detectable (i.e.,the detectable clusters emitted a sufficient amount of fluorescence topropagate through the filter layer of the biosensor and/or the emittedfluorescence was not negatively affected by crosstalk such that theclusters could not be differentiated). The number of sequencing cycles(e.g., incorporations of nucleotides to the sstDNA) was 151 cycles. Thesequencing data from sixteen (16) tiles from the flowcell were analyzed.

Table 1 provided below provides data from the operating sessiondescribed above, which include 151 cycles in the sequencing run oroperational session. Table 1 includes the total number of kilobasesdetected during the operating session from all tiles. Table 1 alsoprovides information provided, on average, for all tiles from theflowcell. Notably, the mismatch rate for clusters on the flowcell thatprovided optical signals that passed the filter layer was 0.94%+/−0.31%.FIG. 37 is a graph illustrating the mismatch rate per cycle for one ofthe tiles. Table 2 is also provided below and provides similar data foranother operating session, which was performed before the operatingsession of Table 1. The mismatch rate in Table 2 is 1.31%+/−0.75%.Further, both Table 1 and 2 show that around 75% of the PhiX sequencealigned to the known PhiX sequence. As such, the biosensor was effectivein detecting the optical signals to determine the sequence of the PhiXDNA during the SBS sequencing protocol.

TABLE 1 Tile Mean ± SD for Lane 1 Lane % Info 1^(st) intensity LaneCycle after 20 % 1% Yield Clusters Clusters Int cycles % PF AlignAlignment Mismatch (kbases) (raw) (PF) (PF) (PF) Clusters (PF) Score(PF) Rate (PF) 120025 54695 ± 49680 ± 377.8 ± 96.4 ± 86.59 ± 75.02 ±1.48 ± 0.94 ± 15628 17030 141.8 7.8 17.86 21.28 0.38 0.31

TABLE 2 Tile Mean ± SD for Lane 1 Lane % Info 1st intensity Lane Cycleafter 20 % 1% Yield Clusters Clusters Int cycles % PF Align AlignmentMismatch (kbases) (raw) (PF) (PF) (PF) Clusters (PF) Score (PF) Rate(PF) 126656 45619 ± 35819 ± 1498.9 ± 88.3 ± 74.51 ± 73.89 ± 51.69 ± 1.31± 10159 15403 447.0 3.3 25.86 30.13 14.63 0.75

Accordingly, in one embodiment, a biosensor is provided, such as thebiosensor 400. The biosensor may include a flow cell and a detectiondevice that includes a plurality of stacked substrate layers. Thedetection device has a detector surface that is configured to supportreaction sites. The stacked layers include a filter layer and asolid-state imager coupled to the filter layer. The filter layerincludes filter walls and a light-absorbing material that is depositedbetween adjacent filter walls. The light-absorbing material isconfigured to prevent transmission of excitation light and permittransmission of emission signals, wherein the adjacent filter wallsdefine a detection path therebetween through the correspondinglight-absorbing material toward the solid-state imager. The filter wallsmay be configured to reflect the emission signals. The flow cell ismounted to the detector surface and defines a flow channel between atleast one surface of the flow cell and the detection device. The atleast one surface of the flow cell includes a material that permitstransmission of the excitation light.

In one aspect, the solid-state imager includes a CMOS image sensorcomprising an array of light detectors that are configured to detect theemission signals. In some embodiments, each of the light detectors hasonly a single pixel and wherein a ratio of the pixels to the detectionpaths defined by the filter walls is substantially one-to-one.

In another aspect, a length of the detection path is less than 10microns.

In another aspect, the filter walls substantially define an image areaalong the detector surface. The reaction site being located within theimage area and having a site area that is less than the image area.

In another aspect, the stacked layers are formed in a layer by layermanner using integrated circuit processing technologies.

In another aspect, the filter walls include first and second interiorportions. The first interior portion extends in a direction from thereaction sites toward the solid-state imager. The second interiorportion extends in a direction from the first interior portion towardthe solid-state imager. The first and second interior portions havedifferent reflectivity.

In another aspect, the filter walls include first and second interiorportions. The first interior portion extends in a direction from thereaction sites toward the solid-state imager. The second interiorportion extends in a direction from the first interior portion towardthe solid-state imager. The second interior portion is configured toattenuate the emission signals from the reaction sites more than thefirst interior portion.

In another aspect, the detection path has a width that decreases as thedetection path extends in a direction from the reaction sites toward thesolid-state imager.

In another aspect, a ratio between a height of the filter walls and awidth of the detection path that extends between the adjacent filterwalls is at least 2.5:1.

In another aspect, the reaction sites include discrete metal regions onthe detector surface.

In another aspect, the reaction sites include discrete clusters ofnucleic acids.

In another embodiment, a method of analyzing signal data from abiosensor including a detection device is provided. The detection deviceincludes an array of light detectors. Each of the light detectors isassociated with at least one reaction site. The reaction sites includeanalytes-of-interest. The method includes (a) obtaining the signal datafrom the light detectors, the signal data including light scores thatare based on an amount of light detected by the light detectors during aplurality of imaging events; (b) analyzing the light scores from a groupof light detectors for each of the plurality of the imaging events; (c)determining respective crosstalk functions of the light detectors in thegroup, each of the crosstalk functions for a corresponding lightdetector being based on the amount of light detected by other lightdetectors in the group; and (d) analyzing the signal data for each ofthe imaging events using the crosstalk functions to determinecharacteristics of the analytes-of-interest.

In one aspect, the analyzing the light scores includes comparing thelight scores of adjacent reaction sites to a predetermined score valueto determine whether the adjacent reaction sites provided emissionsignals.

In another aspect, the crosstalk function is based on a number ofadjacent reaction sites that provide emission signals and locations ofthe adjacent reaction sites that provide the emission signals.

In another aspect, the light scores correspond to voltage signals.

In another aspect, the light detected by the light sensors includesfluorescence emission signals. The fluorescent emission signals may beprovided in response to an excitation event.

In another aspect, the imaging events occur according to a predeterminedprotocol.

In another aspect, the reaction sites include discrete metal regions onthe detector surface.

In another aspect, the reaction sites include discrete clusters ofnucleic acids.

In another embodiment, a system for biological and/or chemical analysisis provided. The system includes a receptacle that is configured toreceive and establish electrical and fluidic connections with abiosensor. The biosensor is configured to have an array of lightdetectors in which each of the light detectors is associated with atleast one reaction site located on a detector surface. The reactionsites are configured to include analytes-of-interest. The system alsoincludes a fluidic control system for controlling a flow of fluidthrough the biosensor along the detector surface. The fluidic controlsystem includes an upstream conduit for providing the fluid to thebiosensor and a downstream conduit for removing the fluid. The systemalso includes an illumination system that is configured to directexcitation light toward the biosensor to illuminate the reaction sites,wherein at least some of the reaction sites provide emission signalswhen illuminated. The system also includes a system controller includingan analysis module. The analysis module is configured to obtain signaldata from the light detectors. The signal data includes light scoresthat are based on an amount of light detected by the light detectorsduring a plurality of imaging events. The system controller is alsoconfigured to analyze the light scores from a group of light detectorsfor each of the plurality of the imaging events. The system controlleris also configured to determine respective crosstalk functions of thelight detectors in the group. Each of the crosstalk functions for acorresponding light detector is based on the amount of light detected byother light detectors in the group. The system controller is alsoconfigured to analyze the signal data for each of the imaging eventsusing the crosstalk functions to determine characteristics of theanalytes-of-interest.

In one aspect, the analyzing the light scores includes comparing thelight scores of adjacent reaction sites to a predetermined score valueto determine whether the adjacent reaction sites provided emissionsignals.

In another aspect, the crosstalk function is based on a number ofadjacent reaction sites that provided emission signals and locations ofthe adjacent reaction sites that provided the emission signals.

In another aspect, the emission signals are provided in response to anexcitation event.

In another aspect, the system controller controls operation of thefluidic control system and the illumination system according to apredetermined protocol to provide the imaging events. The predeterminedprotocol may be a sequencing-by-synthesis protocol.

In another embodiment, a method of manufacturing a biosensor isprovided. The method includes providing a solid-state imager includingan array of light detectors. The method also includes coupling a filterlayer to the solid-state imager. The filter layer includes filter wallsand a light-absorbing material that is between adjacent filter walls.The light-absorbing material is configured to prevent transmission ofexcitation light and permit transmission of emission signals. Theadjacent filter walls define a detection path therebetween through thecorresponding light-absorbing material toward the solid-state imager.The filter walls are configured to reflect the emission signals. Themethod also includes coupling a passivation layer to the filter layer.The passivation layer has a detector surface. The method also includespositioning a flow cell over the detector surface. The flow cell definesa flow channel between at least one surface of the flow cell and thedetector surface.

In one aspect, the coupling the filter layer operation includes couplinga substrate material to the solid-state imager and etching the substratematerial to define chambers before the passivation layer is coupled.

In another aspect, the method further includes depositing alight-absorbing material into the chambers.

In another aspect, the method further comprises applying a coating towalls that define the chambers. The coating may be more reflective thanmaterial that defines the walls.

In another aspect, the method also includes preparing reaction sitesalong the detector surface.

In another aspect, the preparing operation includes locating thereaction sites at designated locations along the detector surface.

It is to be understood that the above description is intended to beillustrative, and not restrictive. For example, the above-describedembodiments (and/or aspects thereof) may be used in combination witheach other. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from its scope. While the specific components andprocesses described herein are intended to define the parameters of thevarious embodiments of the invention, they are by no means limiting andare exemplary embodiments. Many other embodiments will be apparent tothose of skill in the art upon reviewing the above description. Thescope of the invention should, therefore, be determined with referenceto the appended claims, along with the full scope of equivalents towhich such claims are entitled. In the appended claims, the terms“including” and “in which” are used as the plain-English equivalents ofthe respective terms “comprising” and “wherein.” Moreover, in thefollowing claims, the terms “first,” “second,” and “third,” etc. areused merely as labels, and are not intended to impose numericalrequirements on their objects. Further, the limitations of the followingclaims are not written in means-plus-function format and are notintended to be interpreted based on 35 U.S.C. § 112, sixth paragraph,unless and until such claim limitations expressly use the phrase “meansfor” followed by a statement of function void of further structure.

What is claimed is:
 1. A method comprising: illuminating a plurality ofreaction sites of a detection device with excitation light, where thedetection device comprises a plurality of stacked layers under theplurality of reaction sites, where at least some of the reaction siteshave one or more analytes-of-interest, the stacked layers including afilter layer and a solid-state imager coupled to the filter layer, wherethe solid state imager comprises light detectors, the filter layercomprising filter walls and a light-absorbing material that is depositedbetween adjacent filter walls, the light-absorbing material configuredto prevent transmission of excitation light and permit transmission oflight emissions, wherein the adjacent filter walls define a detectionpath therebetween through the corresponding light-absorbing materialtoward the solid-state imager, the filter walls configured to reflectthe light emissions toward the light detectors, and the filter wallsinclude first and second interior portions, the first interior portionextending in a direction from the reaction sites toward the lightdetectors, the second interior portion extending in a direction from thefirst interior portion toward the light detectors; and detecting thelight emissions by the light detectors.
 2. The method of claim 1,wherein at least a portion of the light emissions detected by the lightdetectors include crosstalk light emissions from a non-associatedreaction site.
 3. The method of claim 2, further comprising: obtainingthe signal data from the light detectors, the signal data includinglight scores that are based on an amount of the light emissions detectedby the light detectors during a plurality of imaging events; analyzingthe light scores from a group of light detectors for each of theplurality of the imaging events; determining respective crosstalkfunctions of the light detectors in the group from the analyzing thelight scores from the group of light detectors for each of the pluralityof the imaging events, wherein each of the crosstalk functions for acorresponding light detector in the group is based on the amount of thelight emissions detected by other light detectors in the group; andanalyzing the signal data for each of the imaging events using thecrosstalk functions to determine characteristics of theanalytes-of-interest.
 4. The method of claim 3, wherein the determiningrespective crosstalk functions of the light detectors in the groupincludes: for each imaging event in the plurality of imaging events:determining a light score for each of the light detectors in the groupfrom the imaging event; identifying, as emitting, the at least onereaction site associated with the respective light detectors in thegroup having the light score for the respective imaging event greaterthan or equal to a predetermined score value; identifying, asnon-emitting, the reaction sites associated with the respective lightdetectors in the group having the light score for the respective imagingevent less than the predetermined score value; determining that thelight score of the light detectors associated with non-emitting reactionsites is entirely from crosstalk emissions; and determining, for each ofthe non-emitting reaction sites, the number of adjacent reaction sitesidentified as emitting and the position of each adjacent reaction siterelative to each non-emitting reaction site; and comparing the lightscores of non-emitting reaction sites from the plurality of imagingevents to determine crosstalk portions of the light scores provided bythe adjacent reaction sites.
 5. The method of claim 3, wherein each ofthe crosstalk functions of the light detectors in the group is based ona number of adjacent reaction sites that provided the light emissionsand locations of the adjacent reaction sites that provided the lightemissions.
 6. The method of claim 1, wherein the analytes-of-interestare nucleic acids.
 7. The method of claim 3, wherein the light scorescorrespond to voltage signals.
 8. The method of claim 1, wherein thelight emissions detected by the light detectors includes fluorescenceemission signals.
 9. The method of claim 1, wherein the reaction sitesinclude discrete metal regions on the detector surface having discreteclusters of nucleic acids.
 10. The method of claim 1, wherein thereaction sites include discrete metal regions on the detector surface.11. The method of claim 1, wherein the reaction sites include discreteclusters of nucleic acids.
 12. The method of claim 1, wherein a densityof the reaction sites is at least one million reaction sites per squaremillimeter.
 13. The method of claim 1, wherein the light detectors havedetection areas that are less than about 50 μm².
 14. The method of claim3, wherein the analytes-of-interest are nucleic acids that form clustersat the reaction sites and the plurality of imaging events occur inaccordance with a sequencing-by-synthesis protocol in whichfluorescently-labeled nucleotides are incorporated into the nucleicacids prior to each imaging event.
 15. The method of claim 1, whereinthe detection device includes a passivation layer that defines thedetector surface, the analytes-of-interest being immobilized to thedetector surface and exposed to a flow channel above the detectorsurface, the light emissions propagating through the passivation layertoward the light detectors.
 16. The method of claim 15, wherein thepassivation layer extends between the filter layer and the flow channel,and at least some of the crosstalk light emissions propagate through theflow channel and the passivation layer and over one or more of thefilter walls.
 17. The method of claim 15, wherein the passivation layerextends between the filter layer and the flow channel; thelight-absorbing material filters the excitation light; and the crosstalklight emissions propagate through the flow channel, the passivationlayer, and the light-absorbing material to the light detectors.
 18. Themethod of claim 1, wherein the second interior portion is to attenuatethe light emissions from the reaction sites more than the first interiorportion.
 19. The method of claim 1, wherein the first interior portionis coated with a reflective coating material and the second interiorportion is not coated or is coated with a different material; and thelight emissions from the reaction sites that are reflected by thecoating are less attenuated than the light emissions that are reflectedby the second interior portion of the filter walls.
 20. The method ofclaim 1, wherein a width for each of the detection paths decreases asthe detection path extends toward the light detector; a thickness of thefilter wall begins to increase as the filter wall transitions from thefirst interior portion to the second interior portion; and as thethickness of the filter wall increases, the width for the respectivedetection path decreases.